WO2013175164A1

WO2013175164A1 - Characterization, classification and identification of microorganisms

Info

Publication number: WO2013175164A1
Application number: PCT/GB2013/000239
Authority: WO
Inventors: Colin P. GODDARD; David Hugh Williams; Arthur Keith Turner; John Wain
Original assignee: Discuva Limited
Priority date: 2012-05-24
Filing date: 2013-05-23
Publication date: 2013-11-28

Abstract

Disclosed is a method for identifying a test microorganism comprising the steps of: (a) sequencing complete or partial genomic DNA from the test microorganism; (b) identifying repeated nucleotide n-mer sequences present at a copy number≥r, and (c) comparing the repeated nucleotide n-mer sequences identified in step (b) with those of one or more reference microorganism(s).

Description

CHARACTERIZATION, CLASSIFICATION AND IDENTIFICATION OF

MICROORGANISMS

Field of the Invention

The present invention relates to methods for characterizing microorganisms (including bacteria, viruses and fungi) for various purposes, including classification, identification, diagnosis and sub-typing for epidemiology and virulence determination. The methods involve sequencing complete or partial genomic DNA from a test microorganism and identifying repeated nucleotide n-mer sequences present at a copy number≥r. The repeated nucleotide n-mer sequences are then compared with those present in one or more reference microorganism(s). The results of the comparison can be used to characterize, classify or identify the test microorganism. The methods of the invention find application in the identification of bacterial pathogens in biological samples, including mixed cultures, and complex communities derived from clinical samples for the diagnosis of infectious diseases and other purposes,

Background to the Invention Conventional determinative bacteriology relies largely on the characterization of phenotypic properties of pure cultures obtained from specimens after cultivation and isolation of bacteria on appropriate, usually selective, laboratory media. Newer phenotypic methods using mass spectrometry still depend upon purified isolates and discrimination is at the same level as the 16S methods described below.

More accurate identification, typing and sub-typing traditionally uses extended biochemical tests involving growth on a range of substrates, serotyping with antibodies (typically raised in rabbits) against specific antigens on the surface of the microorganism or differential lysis by bacteriophage. Such methods are time-consuming, labour-intensive and the convergence of phenotypic traits among unrelated bacteria can complicate analysis and lead to errors.

The ever-increasing amount of sequence data from microorganisms, including partial sequences, targeted sequencing, sample sequencing and whole genome sequence data generated by new or next generation sequencing methods (NGS, being rapid or high throughput methods used in place of Sanger sequencing), has made various molecular approaches more tractable. Common examples of such approaches include comparative sequencing of PCR-amplified 16S ribosomal RNA genes (rDNA), isotopic or fluorescently labelled hybridization probes (molecular beacons), or reverse transcription of ribosomal RNA (rRNA) and amplification (RT-PCR, or "Eberwine-type" amplification) used in conjunction with hybridization probes or sequencing. Currently, 16S rRNA or the genes thereof (rDNA) comprise the largest set of gene-specific sequence data. However, relevant information for other targets including CRISPAs (spacer regions in between large, often non-perfect repeats, such as spoligotyping typing for Mycobacterium tuberculosis), Multi Locus Variable Number Tandem repeat (VNTR) Analysis (MLVA), (including MIRU typing for Mycobacterium tuberculosis, 5S rRNA, 23S rRNA, rRNA spacer regions, RNase P RNA, housekeeping genes used for multi-locus sequence typing (MLST) etc is also accumulating rapidly, in part because of complete genome sequencing efforts. For example MLVA is carried out by estimating the number of defined repeats, in a tandem chain; this is usually carried out by size analysis of PCR products but long read sequencing of the large regions involved is becoming more accepted. NGS is not used because the size of the repeat chains is too large. Existing sequence-based approaches are limited by problems arising from gene-target selection. For example, members of the Streptococcus mitis group, which include

Streptococcus pneumoniae, have indistinguishable 16S rRNA gene sequences and many bacterial species do not have sufficient variation in the classic VNTR loci. Moreover, accurate identification requires high-quality, comprehensive reference libraries.

It has now been realised that there are inherent limitations to sequence-based microbial identification based on particular targets (e.g. 16S rRNA and VNTR loci) and that these problems, arising from sequence errors and read length in both test organism and sequence databases, can be overcome by characterizing test microorganisms on the basis of genome wide repeated oligomers nucleic acid sequences defined by: (a) the length of the repeated oligomeric nucleotide sequence (n-mer); and (b) the copy number threshold r of the repeated nucleotide n-mer sequences.

The present inventors have discovered that the use of repeated sequence motifs provides a very accessible readout of "relatedness" which is tolerant of short read sequence data, mismatch and sequencing errors. Changing the length of the n-mer or the minimum copy number r permits the resolution of the technique to be adjusted, permitting identification at the level of genus, species or strain as well as the construction of dendograms showing overall relatedness between test microorganism(s) and a number of reference

species/strains/subtypes/isolates.

The identity (nucleotide sequence) and frequency of the repeated n-mer sequences is potentially unique (and so distinctive for particular species or strains). The gap distance between n-mers can also be exploited in identification of species and sub-species (strain) recognition by allowing the calculation of frequency and so allowing comparison of the numbers of each unique repeat within the genome.

The invention therefore finds particular application in clinical diagnostic technology (using collated reference genomes) from both isolates and sequence data generated directly from clinical specimens.

Summary of the Invention

In a first aspect of the present invention, there is provided a method for identifying a test microorganism comprising the steps of:

(a) sequencing complete or partial genomic DNA from the test microorganism;

(b) identifying repeated nucleotide n-mer sequences present at a copy number≥r, and

(c) comparing the repeated nucleotide n-mer sequences identified in step (b) with those of one or more reference microorganism(s).

In another aspect, there is provided a method for characterizing a microorganism comprising the steps of: (a) sequencing complete or partial genomic DNA from the microorganism;

(b) identifying repeated nucleotide n-mer sequences present at a copy number≥r, and optionally

(c) determining the gap length between repeated nucleotide n-mer sequence

sequences identified in step (b); and/or

(d) estimating the total number of n-mers in the genome directly. In step (c) (above), the gap length may be used to derive the frequency of the n-mer (e.g. number per kilobase) and/or the pattern of frequencies for several n-mers.

Frequency determinations find particular application in embodiments where partial genomic DNA is sequenced in step (a).

In another aspect, there is provided a computer system for use in a method as defined in any one of the preceding claims comprising: (a) a test microorganism database comprising the sequence and copy number of repeated nucleotide n-mer sequences present at a copy number≥r, and (b) a reference database of complete or partial genomic DNA sequences of a plurality of reference microorganisms.

The reference database may be in-house, one of many commercially available databases or publical (e.g. part of library websites or open access research facilities or from service laboratories accredited for the generation of accredited data for human, animal medical proposes or the food industry or industrial manufacture etc.).

The threshold copy number is preferably greater than 10, but may be selected to vary the resolution of the method. Accordingly, r may be chosen according to the needs and preferences of the user, so that the threshold copy number may be 5, 10, 15, 20, 25, 30, 35, 40 or greater than 40.

The length of the repeated nucleotide sequence is preferably about 12 (e.g. 12), but may be selected to vary the resolution of the method. Accordingly, n may be chosen according to the needs and preferences of the user, so that the length of the repeated nucleotide may be 6-24, 8-16 or 10-14.

The methods of the invention find utility in a wide range of applications, including clinical diagnostics, epidemiology, and food microbiology. The methods are particularly suited to the direct analysis of samples (including clinical samples), where complete or partial genomic DNA from microorganisms present in a sample is extracted and analysed without a preliminary culturing step (so greatly reducing the time needed for e.g. clinical diagnosis).

Other aspects of the invention are as defined in the claims attached hereto. Detailed Description of the Invention

Definitions and general preferences Where used herein and unless specifically indicated otherwise, the following terms are intended to have the following meanings in addition to any broader {or narrower) meanings the terms might enjoy in the art:

Unless otherwise required by context, the use herein of the singular is to be read to include the plural and vice versa. The term "a" or "an" used in relation to an entity is to be read to refer to one or more of that entity. As such, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

As used herein, the term "comprise," or variations thereof such as "comprises" or

"comprising," are to be read to indicate the inclusion of any recited integer (e.g. a feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method/process steps or limitations) but not the exclusion of any other integer or group of integers. Thus, as used herein the term "comprising" is inclusive or open-ended and does not exclude additional, unrecited integers or method/process steps.

The phrase "consisting essentially of" is used herein to require the specified integer(s) or steps as well as those which do not materially affect the character or function of the claimed invention.

As used herein, the term "consisting" is used to indicate the presence of the recited integer (e.g. a feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method/process steps or limitations) alone.

The terms Gram-negative bacterium and Gram-positive bacterium are terms of art defining two distinct classes of bacteria on the basis of certain cell wall staining characteristics.

The term low G+C Gram-positive bacterium is a term of art defining a particular subclass of evolutionary related bacteria within the Gram-positives on the basis of the composition of the bases in the DNA. The subclass includes Streptococcus spp., Staphylococcus spp., Listeria spp., Bacillus spp., Clostridium spp., Enterococcus spp. and Lactobacillus spp ).

The term high G+C Gram-positive bacterium is a term of art defining a particular subclass of evolutionarily related bacteria within the Gram-positives on the basis of the composition of the bases in the DNA. The subclass includes actinomycetes (actinobacteria) including Actinomyces spp., Arthrobacter spp., Corynebacterium spp., Frankia spp., Micrococcus spp., Micromonospora spp., Mycobacterium spp., Nocardia spp., Propionibacterium spp. and Streptomyces spp.

Exemplary DNA sequencing methods for use according to the invention

Any suitable high-throughput sequencing technique can be used for sequencing the genomic DNA of the test microorganism, and there are many commercially available sequencing platforms that are suitable for use in the methods of the invention and some in development but not yet in use. Sequencing-by-synthesis (SBS)-based sequencing platforms are particularly suitable for use in the methods of the invention: for example, the lllumina™ system generates millions of relatively short sequence reads (54, 75 or 100bp) and is particularly preferred. Other suitable techniques include methods based on reversible dye-terminators.

Exemplary bacterial targets of the methods of the invention

The methods and systems of the invention find application in the characterization, classification or identification of any microorganism, including bacteria, viruses and fungi. Preferred are methods and systems applied to the characterisation, classification or identification of bacteria, and especially pathogenic bacteria of clinical importance.

Thus, the methods and systems of the invention may be applied to the characterization, classification or identification of: (a) Gram-positive, Gram-negative and/or Gram-variable bacteria; (b) spore-forming bacteria; (c) non-spore forming bacteria; (d) filamentous bacteria; (e) intracellular bacteria; (f) obligate aerobes; (g) obligate anaerobes; (h) facultative anaerobes; (i) microaerophilic bacteria and/or (f) opportunistic bacterial pathogens. In certain embodiments, the invention is used in the characterization, classification or identification of one or more bacteria of the following genera: Acinetobacter (e.g. A.

baumannii); Aeromonas (e.g. A. hydrophila); Bacillus (e.g. B. anthracis); Bacteroides (e.g.

B. fragilis); Bordetel!a (e.g. B. pertussis); Borrelia (e.g. B. burgdorferi); Brucella (e.g. B. abortus, B. can/s, B. melitensis and B. suis); Burkholderia (e.g. B. cepacia complex);

Campylobacter (e.g. C. jejuni); Chlamydia (e.g. C. trachomatis, C. suis and C. muridarum); Chlamydophila (e.g. (e.g. C. pneumoniae, C. pecorum, C. psittaci, C. abortus, C. felis and

C. caviae); Citrobacter (e.g. C. freundii); Clostridium (e.g. C. botulinum, C. difficile, C. perfringens and C. tetani); Corynebacterium (e.g. C. diphteriae and C. glutamicum);

Enterobacter(e.g. E. cloacae and E. aerogenes); Enterococcus (e.g. E. faecalis and E. faecium); Escherichia (e.g. E. coli); Flavobacterium; Francisella (e.g. F. tularensis);

Fusobactehum (e.g. F. necrophorum); Haemophilus (e.g. H. somnus, H. influenzae and H. parainfluenzae); Helicobacter (e.g. H. pylori); Klebsiella (e.g. K. oxytoca and K.

pneumoniae), Legionella (e.g. L. pneumophila); Leptospira (e.g. L interrogans); Listeria (e.g. L. monocytogenes); Moraxella (e.g. M. catarrhalis); Morganella (e.g. M. morganii);

Mycobacterium (e.g. M. leprae and M. tuberculosis); Mycoplasma (e.g. M. pneumoniae);

Neisseria (e.g. N. gonorrhoeae and N. meningitidis); Pasteurella (e.g. P. multocida);

Peptostreptococcus; Prevotella; Proteus (e.g. P. mirabiHs and P. vulgaris), Pseudomohas

(e.g. P. aeruginosa); Rickettsia (e.g. P. rickettsii); Salmonella (e.g. S. typhi and S.

typhimurium); Serratia (e.g. S. marcesens); Shigella (e.g. S. flexnaria, S. dysenteriae and

S. sonnei); Staphylococcus (e.g. S. aureus, S. haemolyticus, S. intermedius, S. epidermidis and S. saprophyticus); Stenotrophomonas (e.g. S. maltophila); Streptococcus (e.g. S. agalactiae, S. mutans, S. pneumoniae and S. pyogenes); Treponema (e.g. T. pallidum);

Vibrio (e.g. V. cholerae) and Yersinia (e.g. Y. pestis).

The invention may be used in the characterization, classification or identification of multidrug resistant bacteria, including, but not limited to penicillin-resistant, methicillin-resistant, quinolone- resistant, macrolide-resistant, and/or vancomycin-resistant bacterial strains, including for example penicillin-, methicillin-, macrolide-, vancomycin-, and/or quinolone- resistant Streptococcus pneumoniae; penicillin-, methicillin-, macrolide-, vancomycin-, and/or quinolone-resistant Sfaphy/ococcus aureus; penicillin-, methicillin-, macrolide-, vancomycin-, and/or quinolone-resistant Streptococcus pyogenes; and penicillin-, methicillin- , macrolide-, vancomycin-, and/or quinolone-resistant enterococci. Thus, the invention may also be used to target strains of Staphylococcus aureus (SA) which are usually multidrug resistant (MR), for example selected from any of C-MSRA1 , C- MRSA2, C-MRSA3, C-MSRA4, Belgian MRSA, Swiss MRSA and any of the EMRSA strains.

The invention may be used in the characterization, classification or identification of high G+C Gram-positive bacteria. The term "high G+C Gram-positive bacteria" is a term of art defining a particular class of evolutionarily related bacteria. The class includes Micrococcus spp. (e.g. M. luteus), Mycobacterium spp. (for example a fast- or slow-growing

mycobacterium, e.g. M. tuberculosis, M. leprae, M. smegmatis or M. bovis), Streptomyces spp. (e.g. S. rimosus and S. coelicolor) and Corynebacterium spp. (e.g. C. glutamicum).

The invention may be used in the characterization, classification or identification of low G+C Gram-positive bacteria. The term "low G+C Gram-positive bacteria" is a term of art defining a particular class of evolutionarily related bacteria. The class includes members of the Firmicutes phylum, including for example Staphylococcus spp. and Bacillus spp.

Exemplification The invention will now be described with reference to specific Examples. These are merely exemplary and for illustrative purposes only: they are not intended to be limiting in any way to the scope of the monopoly claimed or to the invention described. These examples constitute the best mode currently contemplated for practicing the invention. Example 1

All nucleotide 12-mer sequences (12 base long sub-sequences) from a genomic sequence derived from a test microorganism are sorted into alphabetical order. This may be achieved by any suitable algorithm, for example a Radix-type sort.

When a 12-mer is found that is repeated and the number of repeats exceeds a set threshold, the 12-mer sequence and its repeat count are stored. At the end of the sorting process, the repeat statistics are printed to an output file for the test genome. The process is then repeated, and separate output files generated, for all genomes of interest. The total number of repeats, in this case, represents the frequency of repeats because all genomes are of equivalent size. For genomes of variable size (or where partial genome sequences are used), frequencies rather than absolute numbers may be used.

An all-against-all comparison is then performed on the output files generated as described above. The repeats from a specific test genome are compared with the repeats in all other genomes. A score is compiled for the repeats from the test genome against the other genomes where the same repeated sequence occurs.

The output below shows the results when the test genome Acinetobacter baumannii AB0057, is compared to a range of other bacterial genomes from Klebsiella pneumoniae, Escherichia coli, Neisseria gonorrhoeae, S. Typhimurium, S. Typhi strains and other Acinetobacter baumannii strains.

The number on the left show the count of all repeated sequences shared by the test strain and each of the compared strains. The higher the number, the more the compared genomes have in common.

Acinetobacter baumannii AB0057

1493, Acinetobacter baumannii AB0057

385, Acinetobacter baumannii AYE

367, Acinetobacter baumannii AB307-0294

6, Klebsiella pneumoniae 342

4, Klebsiella pneumoniae subsp. pneumoniae HS11286

4, Escherichia coli APEC 01

3, Escherichia coli 536

3, Neisseria gonorrhoeae NCCP11945

3, Typhimurium str. T000240

2, Typhi str. CT18

2, Typhimurium str. D23580

2, Escherichia coli 55989

2, Typhimurium str. UK-1

1 , Typhi str. P-stx-12

1 , Pseudomonas aeruginosa PA7

1 , Typhi str. Ty2 The process is then repeated for all of the other Klebsiella pneumoniae, Escherichia coli, Neisseria gonorrhoeae, Typhimurium, Typhi and Acinetobacter baumannii strains. The full output is shown below:

Acinetobacter baumannii AB0057

1493, Acinetobacter baumannii AB0057

385, Acinetobacter baumannii AYE

367, Acinetobacter baumannii AB307-0294

6, Klebsiella pneumoniae 342

4, Klebsiella pneumoniae subsp. pneumoniae HS11286

4, Escherichia coli APEC 01

3, Escherichia coli 536

3, Neisseria gonorrhoeae NCCP11945

3, Typhimurium str. T000240

2, Typhi str. CT18

2, Typhimurium str. D23580

2, Escherichia coli 55989

2, Typhimurium str. UK-1

1 , Typhi str. P-stx-12

1 , Pseudomonas aeruginosa PA7

1 , Typhi str. Ty2

Acinetobacter baumannii AB307-0294

1689, Acinetobacter baumannii AB307-0294

1548, Acinetobacter baumannii AYE

367, Acinetobacter baumannii AB0057

8, Escherichia coli 536

8, Typhimurium str. T000240

8, Escherichia coli APEC 01

7, Typhimurium str. D23580

7, Escherichia coli 55989

7, Typhimurium str. UK-1

6, Klebsiella pneumoniae subsp. pneumoniae HS11286

3, Neisseria gonorrhoeae NCCP11945

2, Pseudomonas aeruginosa PA7

2, Klebsiella pneumoniae 342 1 , Typhi str. CT18

1 , Typhi str. P-stx-12

1 , Typhi str. Ty2

Acinetobacter baumannii AYE

3314, Acinetobacter baumannii AYE

1548, Acinetobacter baumannii AB307-0294

385, Acinetobacter baumannii AB0057

8, Typhimurium str. Τ0Ό0240

8, Escherichia coli 536

8, Escherichia coli APEC 01

7, Typhimurium str. D23580

7, Escherichia coli 55989

7, Typhimurium str. UK-1

6, Klebsiella pneumoniae subsp. pneumoniae HS11286 4, Neisseria gonorrhoeae NCCP1 1945

2, Pseudomonas aeruginosa PA7

2, Klebsiella pneumoniae 342

1 , Typhi str. P-stx-12

1 , Typhi str. Ty2

Escherichia coli 536

604, Escherichia coli 536

468, Escherichia coli APEC 01

406, Escherichia coli 55989

251 , Klebsiella pneumoniae subsp. pneumoniae HS 11286

233, Klebsiella pneumoniae 342

212, Typhimurium str. T000240

207, Typhimurium str. D23580

207, Typhimurium str. UK-1

204, Pseudomonas aeruginosa PA7

199, Typhi str. CT18

177, Typhi str. P-stx-12

177, Typhi str. Ty2

128, Pseudomonas aeruginosa LESB58

8, Acinetobacter baumannii AYE

8, Acinetobacter baumannii AB307-0294 4, Neisseria gonorrhoeae NCCP11945 3, Acinetobacter baumannii AB0057

2, Neisseria gonorrhoeae FA 1090

Escherichia coli 55989

784, Escherichia coli 55989

419, Escherichia coli APEC 01

406, Escherichia coli 536

286, Klebsiella pneumoniae 342

285, Klebsiella pneumoniae subsp. pneumoniae HS11286

252, Pseudomonas aeruginosa PA7

248, Typhimurium str. T000240

241 , Typhimurium str. D23580

241 , Typhimurium str. UK-1

222, Typhi str. CT18

200, Typhi str. P-stx-12

200, Typhi str. Ty2

149, Pseudomonas aeruginosa LESB58

7, Acinetobacter baumannii AYE

7, Acinetobacter baumannii AB307-0294

6, Neisseria gonorrhoeae NCCP11945

3, Neisseria gonorrhoeae FA 1090

2, Acinetobacter baumannii AB0057

Escherichia coli APEC 01

638, Escherichia coli APEC 01

468, Escherichia coli 536

419, Escherichia coli 55989

264, Klebsiella pneumoniae subsp. pneumoniae HS11286

255, Klebsiella pneumoniae 342

232, Pseudomonas aeruginosa PA7

224, Typhimurium str. T000240

220, Typhimurium str. D23580

220, Typhimurium str. UK-1

208, Typhi str. CT18

191 , Typhi str. P-stx-12

191 , Typhi str. Ty2 143, Pseudomonas aeruginosa LESB58 8, Acinetobacter baumannii AYE

8, Acinetobacter baumannii AB307-0294

4, Acinetobacter baumannii AB0057

3, Neisseria gonorrhoeae NCCP11945

1 , Neisseria gonorrhoeae FA 1090

Klebsiella pneumoniae 342

8745, Klebsiella pneumoniae 342

5681, Pseudomonas aeruginosa PA7

4294, Klebsiella pneumoniae subsp. pneumoniae HS11286

2719, Pseudomonas aeruginosa LESB58

782, Typhimurium str. T000240

768, Typhimurium str. D23580

762, Typhimurium str. UK-1

691 , Typhi str. Ty2

688, Typhi str. P-stx-12

686, Typhi str. CT18

286, Escherichia coli 55989

255, Escherichia coli APEC 01

233, Escherichia coli 536

85, Neisseria gonorrhoeae FA 1090

76, Neisseria gonorrhoeae NCCP11945

6, Acinetobacter baumannii AB0057

2, Acinetobacter baumannii AYE

2, Acinetobacter baumannii AB307-0294

Klebsiella pneumoniae subsp. pneumoniae HS11286

8009, Klebsiella pneumoniae subsp. pneumoniae HS 11286

5484, Pseudomonas aeruginosa PA7

4294, Klebsiella pneumoniae 342

2737, Pseudomonas aeruginosa LESB58

771 , Typhimurium str. T000240

750, Typhimurium str. D23580

746, Typhimurium str. UK-1

660, Typhi str. CT18

652, Typhi str. Ty2 647, Typhi str. P-stx-12

285, Escherichia coli 55989

264, Escherichia coli APEC 01

251 , Escherichia coli 536

84, Neisseria gonorrhoeae FA 1090

81 , Neisseria gonorrhoeae NCCP11945 6, Acinetobacter baumannii AYE

6, Acinetobacter baumannii AB307-0294 4, Acinetobacter baumannii AB0057

Neisseria gonorrhoeae FA 1090

2039, Neisseria gonorrhoeae FA 1090

1189, Neisseria gonorrhoeae NCCP11945 186, Pseudomonas aeruginosa PA7

101 , Pseudomonas aeruginosa LESB58

85, Klebsiella pneumoniae 342

84, Klebsiella pneumoniae subsp. pneumoniae HS11286 41 , Typhimurium str. D23580

41 , Typhimurium str. T000240

41 , Typhimurium str. UK-1

36, Typhi str. P-stx-12

36, Typhi str. CT18

36, Typhi str. Ty2

3, Escherichia coli 55989

2, Escherichia coli 536

1 , Escherichia coli APEC 01

Neisseria gonorrhoeae NCCP11945

1827, Neisseria gonorrhoeae NCCP11945

1189, Neisseria gonorrhoeae FA 1090

181, Pseudomonas aeruginosa PA7

99, Pseudomonas aeruginosa LESB58

81 , Klebsiella pneumoniae subsp. pneumoniae HS11286

76, Klebsiella pneumoniae 342

43, Typhimurium str. D23580

42, Typhimurium str. T000240

42, Typhimurium str. UK-1 34, Typhi str. CT18

32, Typhi str. P-stx-12

32, Typhi str. Ty2

6, Escherichia coli 55989

4, Acinetobacter baumannii AYE

4, Escherichia coli 536

3, Acinetobacter baumannii AB0057

3, Acinetobacter baumannii AB307-0294

3, Escherichia coli APEC 01

Pseudomonas aeruginosa LESB58

9908, Pseudomonas aeruginosa LESB58

9891 , Pseudomonas aeruginosa PA7

2737, Klebsiella pneumoniae subsp. pneumoniae HS11286

2719, Klebsiella pneumoniae 342

493, Typhimurium str. T000240

478, Typhimurium str. D23580

477, Typhimurium str. UK-1

439, Typhi str. Ty2

437, Typhi str. P-stx-12

. 430, Typhi str. CT18

149, Escherichia coli 55989

143, Escherichia coli APEC 01

128, Escherichia coli 536

101, Neisseria gonorrhoeae FA 1090

99, Neisseria gonorrhoeae NCCP11945

Pseudomonas aeruginosa PA7

55494, Pseudomonas aeruginosa PA7

9891 , Pseudomonas aeruginosa LESB58

5681 , Klebsiella pneumoniae 342

5484, Klebsiella pneumoniae subsp. pneumoniae HS11286

734, Typhimurium str. T000240

708, Typhimurium str. D23580

703, Typhimurium str. UK-1

644, Typhi str. Ty2

639, Typhi str. CT18 637, Typhi str. P-stx-12

252, Escherichia coli 55989

232, Escherichia coli APEC 01

204, Escherichia coli 536

186, Neisseria gonorrhoeae FA 1090

181 , Neisseria gonorrhoeae NCCP11945

2, Acinetobacter baumannii AYE

2, Acinetobacter baumannii AB307-0294

1, Acinetobacter baumannii AB0057

Typhi str. CT18

2390, Typhi str. CT18

1759, Typhi str. Ty2

1758, Typhi str. P-stx-12

734, Typhimurium str. T000240

722, Typhimurium str. UK-1

720, Typhimurium str. D23580

686, Klebsiella pneumoniae 342

660, Klebsiella pneumoniae subsp. pneumoniae HS11286

639, Pseudomonas aeruginosa PA7

430, Pseudomonas aeruginosa LESB58

222, Escherichia coli 55989

208, Escherichia coli APEC 01

199, Escherichia coli 536

36, Neisseria gonorrhoeae FA 1090

34, Neisseria gonorrhoeae NCCP11945

2, Acinetobacter baumannii AB0057

1 , Acinetobacter baumannii AB307-0294

Typhi str. P-stx-12

2226, Typhi str. P-stx-12

2216, Typhi str. Ty2

1758, Typhi str. CT18

688, Klebsiella pneumoniae 342

647, Klebsiella pneumoniae subsp. pneumoniae HS11286 637, Pseudomonas aeruginosa PA7

635, Typhimurium str. T000240 630, Typhimurium str. D23580

630, Typhimurium str. UK-1

437, Pseudomonas aeruginosa LESB58

200, Escherichia coli 55989

191 , Escherichia coli APEC 01

177, Escherichia coli 536

36, Neisseria gonorrhoeae FA 1090

32, Neisseria gonorrhoeae NCCP11945

1 , Acinetobacter baumannii AYE

1 , Acinetobacter baumannii AB0057

1 , Acinetobacter baumannii AB307-0294

Typhi str. Ty2

2239, Typhi str. Ty2

2216, Typhi str. P-stx-12

1759, Typhi str. CT18

691 , Klebsiella pneumoniae 342

652, Klebsiella pneumoniae subsp. pneumoniae HS11286 644, Pseudomonas aeruginosa PA7

638, Typhimurium str. T000240

631 , Typhimurium str. D23580

631 , Typhimurium str. UK-1

439, Pseudomonas aeruginosa LESB58

200, Escherichia coli 55989

191 , Escherichia coli APEC 01

177, Escherichia coli 536

36, Neisseria gonorrhoeae FA 1090

32, Neisseria gonorrhoeae NCCP11945

1 , Acinetobacter baumannii AYE

1 , Acinetobacter baumannii AB0057

1 , Acinetobacter baumannii AB307-0294

Typhimurium str. D23580

1250, Typhimurium str. D23580

1223, Typhimurium str. UK-1

1205, Typhimurium str. T000240

768, Klebsiella pneumoniae 342 750, Klebsiella pneumoniae subsp. pneumoniae HS11286

720, Typhi str. CT18

708, Pseudomonas aeruginosa PA7

631 , Typhi str. Ty2

630, Typhi str. P-stx-12

478, Pseudomonas aeruginosa LESB58

241 , Escherichia coli 55989

220, Escherichia coli APEC 01

207, Escherichia coli 536

43, Neisseria gonorrhoeae NCCP11945

41 , Neisseria gonorrhoeae FA 1090

7, Acinetobacter baumannii AYE

7, Acinetobacter baumannii AB307-0294

2, Acinetobacter baumannii AB0057

Typhimurium str. T000240

1299, Typhimurium str. T000240

1217, Typhimurium str. UK-1

1205, Typhimurium str. D23580

782, Klebsiella pneumoniae 342

771 , Klebsiella pneumoniae subsp. pneumoniae HS11286

734, Typhi str. CT18

734, Pseudomonas aeruginosa PA7

638, Typhi str. Ty2

635, Typhi str. P-stx-12

493, Pseudomonas aeruginosa LESB58

248, Escherichia coli 55989

224, Escherichia coli APEC 01

212, Escherichia coli 536

42, Neisseria gonorrhoeae NCCP11945

41, Neisseria gonorrhoeae FA 1090

8, Acinetobacter baumannii AYE

8, Acinetobacter baumannii AB307-0294

3, Acinetobacter baumannii AB0057

Typhimurium str. UK-1

1250, Typhimurium str. UK-1 1223, Typhimurium str. D23580

1217, Typhimurium str. T000240

762, Klebsiella pneumoniae 342

746, Klebsiella pneumoniae subsp. pneumoniae HS11286

722, Typhi str. CT18

703, Pseudomonas aeruginosa PA7

631, Typhi str. Ty2

630, Typhi str. P-stx- 12

477, Pseudomonas aeruginosa LESB58

241, Escherichia coli 55989

220, Escherichia coli APEC 01

207, Escherichia coli 536

42, Neisseria gonorrhoeae NCCP11945

41 , Neisseria gonorrhoeae FA 1090

7, Acinetobacter baumannii AYE

7, Acinetobacter baumannii AB307-0294

2, Acinetobacter baumannii AB0057

Example 2: Identification of different micro-organisms in a mixed culture

To identify different micro-organisms in a mixed culture, the method searches for abundant repeats that are completely specific for a strain or subtype.

Acinetobacter baumannii AB0057 has 1493 12-mer sequences that are repeated 10 or more times. Of these, 385 are shared with Acinetobacter baumannii AYE, only 367 with Acinetobacter baumannii AB307-0294, and 6 or less with each of the other tested strains listed below.

Thus, there are more than 700 repeated sequences that can be used to differentiate Acinetobacter baumannii AB0057 from Acinetobacter baumannii AYE and Acinetobacter baumannii AB307-0294, and more than 1450 repeats that could identify it amongst all of the other tested strains.

Acinetobacter baumannii AB0057

1493, Acinetobacter baumannii AB0057 385, Acinetobacter baumannii AYE

367, Acinetobacter baumannii AB307-0294

6, Klebsiella pneumoniae 342

4, Klebsiella pneumoniae subsp. pneumoniae HS11286

4, Escherichia coli APEC 01

3, Escherichia coli 536

3, Neisseria gonorrhoeae NCCP11945

3, Typhimurium str. T000240

2, Typhi str. CT18

2, Typhimurium str. D23580

2, Escherichia coli 55989

2, Typhimurium str. UK-1

1 , Typhi str. P-stx-12

1 , Pseudomonas aeruginosa PA7

1 , Typhi str. Ty2

Example 3: Repeat hits and repeat counts

The above example shows that Acinetobacter baumannii AB0057 has 1493 12-mers that are repeated 10 or more times, and that 385 of these are shared with Acinetobacter baumannii AYE.

If the number of repeats of these 385 common 12-mers is taken into account, they share a total of 4780 repeated 12-mers. The number of "repeat hits" (385) is the number of distinct 12-mers, repeated 10 or more times, that the two strains have in common. The "repeat count" (4780) is the total number of these repeated 12-mers that the two strains have in common.

Thus, both the number of shared distinct repeated nucleotide n-mer sequences ("repeat h was unknown to the operator) its") and the number of copies of all shared repeated nucleotide n-mer sequences (the "repeat count") are useful statistics in the methods of the invention.

Example 4: Using FASTA data of a completed genome sequence (with any identifying information removed) comparison with high quality genome sequences available from the NCBI site (http://www.ncbi. nlm.nih.gov/genome/) gave the following identification parameters Sequence one: test is Escherichia coli 0104:H4 str. 2011 C-3493

3498, jwTest 1

3443, NC_010468 Escherichia coli ATCC 8739

3422, NC_017625 Escherichia coli

3395, NC_012947 Escherichia coli

3316, NC_016902 Escherichia coli

3222, NC_007779 Escherichia coli

3174, NC_000913 Escherichia coli

3170, NC_012759 Escherichia coli

3158, NC_017638 Escherichia coli

3145, NC 017663 Escherichia coli

3122, NC_017633 Escherichia coli

Sequence two: test is Pseudomonas aeruginosa NCGM2.S1

331660 jwTest 2

254203, NC_002516 Pseudomonas aeruginosa

231687, NCJD09656 Pseudomonas aeruginosa

The operator was blind to the identity of the organism from which the genome data was generated. Thus the invention was able to correctly identify the species from an unknown bacterial sequence and place in order of similarity other members of that species for which an appropriate sequence was available.

Equivalents \

The foregoing description details presently preferred embodiments of the present invention. Numerous modifications and variations in practice thereof are expected to occur to those skilled in the art upon consideration of these descriptions. Those modifications and variations are intended to be encompassed within the claims appended hereto.

Claims

CLAIMS:

1. A method for identifying a test microorganism comprising the steps of: (a) sequencing complete or partial genomic DNA from the test microorganism;

2. The method of claim 1 wherein step (c) comprises counting the number (or determining the frequency) of the repeated nucleotide n-mer sequences identified in step (b) that are shared with one or more reference microorganism(s).

3. The method of claim 2 further comprising the step of identifying the test microorganism on the basis of the degree to which the repeated nucleotide n-mer sequences are shared with the one or more reference microorganism(s).

4. The method of claim 2 or claim 3 wherein in step (c) the number of shared distinct repeated nucleotide n-mer sequences is counted, or the frequency thereof determined.

5. The method of claim 2 or claim 3 wherein in step (c) the number of shared copies of all repeated nucleotide n-mer sequences is counted, or the frequency thereof determined.

6. The method of claim 2 or claim 3 wherein in step (c) both the number of shared distinct repeated nucleotide n-mer sequences and the number of copies of all shared repeated nucleotide n-mer sequences are counted, or the frequencies thereof determined.

7. The method of any one of the preceding claims wherein step (c) comprises determining whether the repeated nucleotide n-mer sequences identified in step (b) include a distinctive repeated nucleotide n-mer sequence that is unique to a particular reference

microorganism.

8. The method of claim 7 further comprising the step of identifying the test microorganism on the basis of the presence of the distinctive repeated nucleotide n-mer sequence identified in step (c).

9. The method of any one of the preceding claims further comprising the step of determining the gap length between said repeated nucleotide n-mer sequences.

10. The method of any one of the preceding claims wherein n = 6-24.

11. The method of claim 10 wherein n = 8-16.

12. The method of claim 11 wherein n = 10-14.

13. The method of claim 12 wherein n is about 12.

14. The method of claim 13 wherein n = 12.

15. The method of any one of the preceding claims wherein r is 10, 15, 20, 25, 30, 35 or 40.

16. The method of any one of the preceding claims for identifying one or more

microorganism(s) present in a mixed culture.

17. The method of claim 16 for identifying two or more microorganism(s) present in mixed culture.

18. The method of any one of the preceding claims wherein the microorganism is from: (a) a clinical isolate; (b) a biological sample, for example a stool sample, blood sample, urine sample, saliva sample or swab; (c) an environmental sample; or (d) a food sample.

19. The method of any one of the preceding claims wherein step (c) comprises comparing the repeated nucleotide n-mer sequences identified in step (b) with those of a plurality of reference microorganisms!

20. The method of claim 19 wherein the plurality of reference microorganisms comprises bacterial pathogens.

21. The method of claim 19 or claim 20 wherein the reference microorganisms are selected from, (a) Gram-positive, Gram-negative and/or Gram-variable bacteria; (b) spore- forming bacteria; (c) non-spore forming bacteria; (d) filamentous bacteria; (e) intracellular bacteria; (f) obligate aerobes; (g) obligate anaerobes; (h) facultative anaerobes; (i) microaerophilic bacteria and/or (f) opportunistic bacterial pathogens.

22. The method of any one of claims 19-21 wherein the reference microorganisms are selected from: Ac/netobacter (e.g. A. baumannii); Aeromonas (e.g. A. hydrophila); Bacillus (e.g. B. anthracis); Bacteroides (e.g. B. fragilis); Bordetella (e.g. B. pertussis); Borrelia (e.g. B. burgdorferi); Brucella (e.g. B. abortus, B. canis, B. melitensis and B. suis); Burkholderia

(e.g. B. cepacia complex); Campylobacter (e.g. C. jejuni); Chlamydia (e.g. C. trachomatis,

C. suis and C. muridarum); Chlamydophila (e.g. (e.g. C. pneumoniae, C. pecorum, C. psittaci, C. abortus, C. felis and C. cav/^'ae); Citrobacter (e.g. C. freundii); Clostridium (e.g.

C. botulinum, C. difficile, C. perfringens and C. tetani); Corynebacterium (e.g. C. diphteriae and C. glutamicum); Enterobacter (e.g. E. cloacae and E. aerogenes); Enterococcus (e.g.

E. faecalis and E. faecium); Escherichia (e.g. E. co!i); Flavobacterium; Francisella (e.g. F. tularensis); Fusobacterium (e.g. F. necrophorum); Haemophilus (e.g. H. somnus, H.

influenzae and H. parainfluenzae); Helicobacter (e.g. H. pylori); Klebsiella (e.g. K. oxytoca and K. pneumoniae), Legionella (e.g. L. pneumophila); Leptospira (e.g. L interrogans); Listeria (e.g. L. monocytogenes); Moraxella (e.g. M. catarrhalis); Morganella (e.g. M.

morganii); Mycobacterium (e.g. M. leprae and M. tuberculosis); Mycoplasma (e.g. M.

pneumoniae); Neisseria (e.g. N. gonorrhoeae and N. meningitidis); Pasteurella (e.g. P. multocida); Peptostreptococcus; Prevotella; Proteus (e.g. P. mirabilis and P vulgaris),

Pseudomonas (e.g. P. aeruginosa); Rickettsia (e.g. R rickettsii); Salmonella (e.g.

serotypes . Typhi and Typhimurium); Serratia (e.g. S. marcesens); Shigella (e.g. S.

flexnaria, S. dysenteriae and S. sonnei); Staphylococcus (e.g. S. aureus, S. haemolyticus,

S. intermedius, S. epidermidis and S. saprophytics); Stenotrophomonas (e.g. S.

maltophila); Streptococcus (e.g. S. agalactiae, S. mutans, S. pneumoniae and S.

pyogenes); Treponema (e.g. Γ. pallidum); Vibrio (e.g. V. cno/erae) and Yersinia (e.g. Y. pestis).

23. A method for characterizing a microorganism comprising the steps of: (a) sequencing complete or partial genomic DNA from the microorganism; (b) identifying repeated nucleotide n-mer sequences present at a copy number≥r, and optionally

(c) determining the gap length between repeated nucleotide n-mer sequence

sequences identified in step (b).

24. A method for classifying or identifying a microorganism comprising characterizing the microorganism according to the method of claim 23.

25. The method of any one of the preceding claims wherein the microorganism is a bacterium.

26. The method of any one of the preceding claims further comprising the step of extracting complete or partial genomic DNA from the test microorganism directly from a sample prior to the sequencing step (a).

27. The method of any one of claims 1-25 further comprising the step of culturing the test microorganism from a sample prior to the sequencing step (a).

28. The method of claim 26 or 27 wherein the sample is selected from: (a) a clinical isolate; (b) a biological or clinical sample, for example a stool sample, blood sample, urine sample, saliva sample or swab; (c) an environmental sample; or (d) a food sample.

29. A method for diagnosing infectious disease comprising the method of any one of the preceding claims.

30. A method for characterising the commensal flora in a human or non-human animal comprising the method of any one of the preceding claims.

31. A method for identifying and sub-typing bacteria comprising the method of any one of the preceding claims.

32. A computer system for use in a method as defined in any one of the preceding claims comprising: (a) a test microorganism database comprising the sequence and copy number of repeated nucleotide n-mer sequences present at a copy number≥r, and

(b) a reference database of complete or partial genomic DNA sequences of a plurality of reference microorganisms.