EP1066407A1 - Procede de differentiation de souches de bacteries - Google Patents

Procede de differentiation de souches de bacteries

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Publication number
EP1066407A1
EP1066407A1 EP98912806A EP98912806A EP1066407A1 EP 1066407 A1 EP1066407 A1 EP 1066407A1 EP 98912806 A EP98912806 A EP 98912806A EP 98912806 A EP98912806 A EP 98912806A EP 1066407 A1 EP1066407 A1 EP 1066407A1
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EP
European Patent Office
Prior art keywords
sequence
direct repeat
primer
spacer
bacterium
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP98912806A
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German (de)
English (en)
Inventor
Johannes Dirk Anthonie Van Embden
Leendert Marinus Schouls
Rudolph Jansen
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SEED CAPITAL INVESTMENTS (SCI-2) BV
Stichting voor de Technische Wetenschappen STW
Original Assignee
SEED CAPITAL INVESTMENTS (SCI-2) BV
Stichting voor de Technische Wetenschappen STW
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Publication of EP1066407A1 publication Critical patent/EP1066407A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria

Definitions

  • the subject invention lies in the field of interstrain differentiation of bacteria.
  • a general method has been developed with which various types of bacteria can be differentiated into separate individual strains. Thus in particular in the clinical setting this method can suitably be used to determine what strain of bacterium is present in a sample. This new method is applicable for discerning between various strains of both Gram negative and Gram positive types of bacteria.
  • the same problems identified for M. tuberculosis are inherent in differentiation of numerous other bacteria. The problems specifically arise for potentially epidemic pathogens and for bacteria that infect hospitals. A more rapid and simple typing method is required. Preferably the testing methods for various bacteria will occur in the same manner ensuring routine use for all types of bacteria for which testing is required. Preferably a test that can be carried out by non specialised personnel using little laboratory space and time is sought after.
  • the method disclosed in W095/31 69 is based on the DNA polymorphism found at a unique chromosomal locus, the "Direct Repeat" (DR) region, which is uniquely present in FI. tuberculosis complex bacteria. This locus was discovered by Hermans et al. (15) in FI.
  • bovis BCG the strain used worldwide to vaccinate against tuberculosis.
  • the DR region in FI. bovis BCG consists of Directly repeated sequences of 36 base pairs, which are interspersed by non-repetitive DNA spacers, each 35 to l base pairs in length (15).
  • the number of copies of the DR sequence in FI. bovis BCG was determined to be 49- In other strains of the FI. tuberculosis complex the number of DR elements was found to vary (15)-
  • the vast majority of the FI. tuberculosis strains contain one or more IS ⁇ llO elements in the DR containing region of the genome.
  • Figure 1 depicts the structure of the DR region of FI. bovis BCG as determined previously by Hermans et al. and Groenen et al. (12, 15).
  • DVR Direct Variant Repeat
  • the DR region is composed of a discrete number of DVR's, each consisting of a constant part (DR) and a variable part (the spacer).
  • the method disclosed in W0 5/31569 is based on a unique method of in vitro amplification of DNA sequences within the DR region and the hybridisation of the amplified DNA with multiple, short synthetic oligomeric DNA sequences based on the sequences of the unique spacer DNA's within the DR region (figure 2). This differs from previous PCR methods in the use of a set of primers with both primers having multiple 3 priming sites as opposed to having one of the primers bind to a fixed priming site such as to a part of IS ⁇ llO. Because FI. tuberculosis complex strains differ in the presence of these spacer sequences, strains can be differentiated by the different hybridisation patterns with a set of various spacer DNA sequences.
  • the method consists of in vitro amplification of nucleic acid using amplification primers in a manner known per se in amplification reactions such as PCR, LCR or NASBA, wherein a pair of primers is used comprising oligonucleotide sequences sufficiently complementary to a part of the Direct Repeat sequence of a microorganism belonging to the FI. tuberculosis complex of microorganisms for hybridisation to a Direct Repeat to occur and subsequently elongation of the hybridized primer to take place, said primer being such that elongation in the amplification reaction occurs for one primer in the 5' Direction and for the other primer in the 3' Direction.
  • a pair of primers comprising oligonucleotide sequences sufficiently complementary to a part of the Direct Repeat sequence of a microorganism belonging to the FI. tuberculosis complex of microorganisms for hybridisation to a Direct Repeat to occur and subsequently elongation of the hybridized primer to take place, said primer being such that
  • the use of such primers implies that all the spacer regions will be amplified in an efficient manner. In particular it is not necessary for extremely long sequences to be produced in order to obtain amplification of spacers located at a distance from the primer. With the instant selection of the primer pairs a heterogenous product is obtained comprising fragments all comprising spacer region nucleic acid. Subsequently the detection of the amplified product can occur simply by using an oligonucleotide probe directed at one or more of the spacer regions one wishes to detect.
  • the primers can have oligonucleotide sequences complementary to non-overlapping parts of the Direct Repeat sequence so that when both primers hybridize to the same Direct Repeat and undergo elongation they will not be hindered by each other.
  • the DRa is selected such that it is complementary to a sequence of the Direct Repeat located to the 5' side of the sequence of the Direct Repeat to which DRb is complementary.
  • the primer used must have an oligonucleotide sequence capable of annealing to the consensus sequence of the Direct Repeat in a manner sufficient for amplification to occur under the circumstances of the particular amplification reaction.
  • a person skilled in the art of amplification reactions will have no difficulty in determining which length and which degree of homology is required for good amplification 4 reactions to occur.
  • the consensus sequence of the Direct Repeat of microorganisms belonging to the FI. tuberculosis complex is given in sequence id. no . 2 and in figure 1.
  • Genera from Group 4 are Legionella (which can cause pneumonia) and Legionnaires disease, the genus Neisseria (of which Neisseria meningitidis is well known as causative agent of meningitis and of which Neisseria gonorrhoeae is another example) , the genus Pseudomonas (renown for hospital infections) and the genus Bordetella (of which Bordetella pertussis is well known as causative agent of whooping cough) .
  • the Enterobacteriacae form a family of 30 genera. These bacteria form a particularly interesting group of Gram negative bacteria that infect humans .
  • Suitable examples of genera from this family are Enterobacter, Escherichia, Shigella, Salmonella, Serratia, Klebsiella and Yersinia.
  • Other less well known pathogenic Enterobacteriacae genera are Cedeca, Citrobacter, Kluyvera, Leclercia, Pantoea, Proteus, Providencia and Hafnia.
  • Other Group 5 families are Pasteurellaceae with the genus Haemophilus and the family Vibrionaceae with the genus Vibrio.
  • Haemophilus influenzae is a leading cause of meningitis in children and also other septicemia conditions.
  • Vibrio cholerae is the causative agent of cholera, V. parahaemolyticus can cause 6 food poisoning and V. vulnificus causes highly fatal septicemia.
  • Shigella is an intestinal pathogen of humans causing bacillary dysentery.
  • Well known strains are S. dysenteriae, S. flexneri, S. boydii, S. sonnei.
  • the genus Salmonella is well known for food poisoning.
  • Well known Salmonella strains are S. typhimurium, S. arizona, S. choleraesuis , S. bongori. Salmonella are also causative agents of typhoid fever, enteric fevers, gastroenteritis and septicemia.
  • the genus Serratia bacteria are opportunistic pathogens for hospitalized humans causing septicemia and urinary tract infections. Examples are S. liquefaciens and S. marcescens. Of the Escherichia E. coli is best known as major cause of urinary tract infections and nosocomial infections including septicemia and meningitis . Other species are usually associated with wound infections. Enterobacter constitutes a problem genus of opportunistic pathogens causing burn wound and urinary tract infections occasionally also meningitis and septicemia. Well known species are E. cloacae, E. sakazakii, E. aerogenes , E. agglomerans, E. gergoviae.
  • Klebsiella are also causative agents of bacteriemia, pneumonia, urinary tact and other human infections in urological, neonatal, intensive care and geriatric patients. Klebsiella pneumoniae and K. oxytoca are examples of species in the genus.
  • Streptococcus and Staphylococcus form examples of such bacteria.
  • Streptococcus pneumoniae, Streptococcus pyogenes and Staphylococcus aureus are examples thereof.
  • the pathogenic bacteria are of interest. These bacteria are dangerous when infecting hospitals in particular.
  • Such organisms comprise Bordetella pertussis and Neisseria menigitidis the causative organism of meningitis is of particular interest. Quite specifically pathogenic bacteria infecting hospitals and bacteria capable of causing epidemics are targets for the differentiation method according to the invention.
  • the invention consists of a method of in vitro amplification of nucleic acid using amplification primers in a manner known per se, in amplification reactions such as PCR, LCR or NASBA, wherein a pair of primers is used comprising oligonucleotide sequences sufficiently 7 complementary to a part of the Direct Repeat sequence of a bacterium other than a microorganism belonging to the M tuberculosis complex of microorganisms for hybridisation to a Direct Repeat to occur and subsequently elongation of the hybridised primer to take place, said primers being such that elongation in the amplification reaction occurs for one primer in the 5 ' Direction and for the other primer in the 3 ' Direction, wherein the Direct Repeat is a sequence with a length between 20-50 base pairs which occurs 5 ⁇ 6 ⁇ times in a contiguous region of the bacterial genome, whereby the Direct Repeat sequences are separated by spacer sequences with a length of between 20-50 nucleotides, said spacer sequences being non repetitive
  • Patscan By using the programme Patscan e.g. on the nucleic acid data bases for microorganism genomic sequences such motifs and thus also the identities of the various species specific Direct Repeats and the corresponding spacer sequences can be obtained.
  • the Direct Repeat can be designated pi with a length between 20-50 basepairs then search for pi 20-50 basepairs downstream of pi.
  • the length of the sequences can be varied as can the intermediate distance and the number of times the Direct Repeat has to occur.
  • a Direct Repeat can often have a length of 30-40 base pairs with a spacer length of 35 ⁇ 45 base pairs.
  • the structures of the DR-like loci in the microorganisms is rather variable (figure 3)-
  • M. tuberculosis the DR locus is large and in most isolates it is disrupted by an insertion element. This is also the case in T. thermophilus, however here the number of DVR's is only 11 and the DR locus is disrupted by two insertion elements.
  • E. coli K12 2 DR loci are present separated by approximately 22kb; in Anabaena the locus is of intermediate size and interrupted by a 130 bp sequence of unknown function or origin.
  • the DR locus is of intermediate size and not disrupted, however there is evidence for a second DR locus on one of the mega plasmids found in this organism.
  • M. jannaschii there is one locus of intermediate size but at several other positions in the genome one or a few other DVR's are found. In most cases the DVR's are linked to a so- called Long Repeat ( R) element of unknown function.
  • R Long Repeat
  • a DR-like sequence was found in the contig 214 of S. pyogenes M1(ATCC 700294) of the genome sequencing project of the University of Oklahoma. Further research into this DR-like sequence in other S. pyogenes revealed spacer polymorphism.
  • the DR regions of eight S. pyogenes isolates were studied. The DR regions were isolated by PCR using primers that were derived from the database (University of Oklahoma, serotype Ml ATCC 700294. The sequence data is available under http://www.genome.ou.edu. This strain contains seven repeats and six spacers . Five of the isolates gave a PCR product, these were a M2 strain, a M4 strain and three Ml strains.
  • the M4 strain contained only a single repeat sequence that was flanked by the same sequences as the ATCC 700294.
  • the M2 strain sequencing did not work, but the size of the PCR fragment indicated that two repeats are present.
  • the three Ml strains were all the same, they contained four repeats and three spacers. The repeats were identical to ATCC 700294 , while one of the spacers was identical to ATCC 700294 and two were different.
  • the Salmonella genomic sequence as sequenced by the University of Washington St Louis has also revealed the presence of DR-like sequences.
  • the DR exhibits high homology with the Direct Repeat of E. coli .
  • One of the contigs revealed 7 Repeats and 6 spacers .
  • a panel of five E. coli isolates and three Shige lla strains were studied. The five E. coli isolates were selected to have an optimal diversity, they were isolated from different species or geographic regions. The Shigella strains are considered separate (sub) species. See Table 1. The isolates were obtained from the collection of Dr. Wim Gaastra.
  • the DR regions were identified by Southern blot of genomic DNA and DRI and DRII regions of E. coli K12. When PCR is indicated the DR regions were identified by the Southern and the PCR. This PCR was done with primers derived from the K12 sequence.
  • the DRI and DRII sequences that could be amplified by PCR were 10 cloned and sequenced. Somehow the DRI regions could not be amplified by PCR using the primers designed on the K12 sequence, while the Southern data demonstrate that DRI is present. Apparently, the recognitions sites for the primers are polymorphic. The sizes of the DRII regions were found to vary greatly between these isolates. The smallest was a single repeat in the S. sonnei strain and the largest was a repeat cluster of at least 15 repeats in E. coli isolate 1008. The sequences of the repeats were highly conserved between these isolates. The S. typhimurium data is obtainable from the Internet http://genome.wustl.edu/gsc/- bacterial/salmonella.html.
  • spacer sequences almost all were unique. Approximately 40 spacers have been sequenced and only three of them were already known from a previously sequenced DR region. This indicates a high number of different spacer sequences in E. coli . Accession number X73 53 provides the Halerofax mediterranei sequence. The sequence can also be found in Molecular Microbiology 17 of 1995 i an article by Mojica et al. (17). The Repeat sequence has also been found in related species .
  • the genomic project of the Methanococcus jannaschii reveals a DR-like sequence as is apparent from the Bult et al article in Science 273 of 1996 (18).
  • the Accession number is U67459 i-a.
  • the invention also covers a method of detection of a bacterium, said bacterium not belonging to the M . tuberculosis complex of microorganisms said method comprising
  • the method can be carried out in a manner such that the hybridisation test is carried out using a number of oligonucleotide probes, said number comprising at least a number of oligonucleotides probes specific for the total spectrum of bacteria it is desired to detect.
  • the oligonucleotide probe is at least seven oligonucleotides long and is a sequence complementary to a sequence selected from any of the spacer sequences of the Direct Repeat region of the bacterium to be determined or is a sequence complementary to fragments or derivatives of said spacer sequences , said oligonucleotide probe being capable of hybridising to such a spacer sequence and comprising at least seven consecutive nucleotides homologous to such a spacer sequence and/or exhibiting at least 60$ homology, preferably exhibiting at least 80$ homology with such a spacer sequence.
  • the method according to the invention is carried out to determine the presence and nature of a pathogenic bacterium selected from the group of Gram negative bacteria of Groups 4 and 5 of Bergeys Determinative Manual of Bacteriology ninth edition.
  • a pathogenic bacterium selected from the group of Gram negative bacteria of Groups 4 and 5 of Bergeys Determinative Manual of Bacteriology ninth edition.
  • Group 5 most specifically the Enterobacteriaceae. Also of interest are the Gram positive bacteria of Group 17- Suitable examples of genera of the pathogenic bacterium to be detected from the group of Gram negative bacteria of Bergeys Determinative Manual of Bacteriology ninth edition are Eschericchia, Shigella, Salmonella, Klebsiella, Enterobacter,
  • Pseudomonas and Bordetella are targets for the differentiation method.
  • the hybridisation pattern is 12 compared with that obtained with a reference.
  • a reference can be the hybridisation pattern obtained with one or more known strains of the bacterium to be determined in analogous manner as the strain to be determined.
  • the reference is a source providing a list of spacer sequences and sources thereof, such as a data bank. Table II exhibits some suitable examples of sequences that occur as Direct Repeat sequences according to the invention for the genera illustrated.
  • a pair of primers according to the invention is a pair wherein both primers comprise oligonucleotide sequences of at least 7 oligonucleotides and are sufficiently complementary to a part of the Direct Repeat sequence of the microorganism E.
  • oligonucleotide sequence comprises at least seven consecutive nucleotides homologous to such a Direct Repeat sequence and/or exhibits at least 60$ homology, preferably at least 80$ homology, most preferably more than 90% homology with the corresponding part of the Direct Repeat sequence.
  • Suitable Direct Repeat sequences are provided in Table II.
  • a primer pair can comprise one primer DRa capable of elongation in the 5' Direction and the other primer DRb capable of elongation in the 3' Direction with DRa being complementary to a sequence of the Direct Repeat located to the 5 ' side of the sequence of the Direct Repeat to which DRb is complementary, the Direct Repeat being present in the Direct Region of E. coli.
  • Another suitable pair comprises primers with oligonucleotide sequences of at least 7 oligonucleotides and are sufficiently complementary to a part of the Direct Repeat sequence of the microorganism S.
  • oligonucleotide sequence comprises at least seven consecutive nucleotides homologous to such a Direct Repeat sequence in particular the Sequence provided in Table II and/or exhibits at least 60$ homology, preferably at least 80$ homology, most preferably more th.-n 90% homology with the corresponding part of the Direct Repeat sequence.
  • such a pair comprises one primer DRa capable of elongation in the 5' Direction and the other primer DRb capable of elongation in the 3' Direction with DRa being complementary to a sequence of the Direct Repeat located to the 5' side of the sequence of the Direct Repeat to which DRb is complementary, the Direct Repeat being present in the Direct Region of S. typhimurium.
  • kits for carrying out a differentiation method according to any 16 of the described embodiments also fall within the scope of the invention.
  • Such kits comprise a primer pair according to any of the described embodiments and optionally an oligonucleotide probe or a carrier, said carrier comprising at least 1 oligonucleotide probe specific for a spacer region of a bacterium to be determined said bacterium not belonging to M tuberculosis complex, preferably the oligonucleotide probe as defined, being an oligonucleocide probe of at least 10 nucleotides, preferably more than 12 nucleotides , in particular comprising between 12 to 40 nucleotides, said probe being sufficiently homologous to any of the spacer sequences or to fragments or derivatives of such spacer sequences to hybridise to such a spacer sequence, said oligonucleotide probe comprising at least 10 consecutive nucleotides homologous to such a spacer sequence and/or exhibiting at least 60% homology,
  • Insertion element IS986 from Mycobacterium tuberculosis a useful tool for diagnosis and epidemiology of tuberculosis. J. Clin. Microbiol. 28:2051-2058.
  • Figure 1 depicts the structure of the DR region of M. bovis BCG as determined previously by Hermans et al. and Groenen et al. (12, 15).
  • DVR Direct Variant Repeat
  • the DR region is composed of a discrete number of DVR's, each consisting of a constant part (DR) and a variable part (the spacer).
  • Figure 2 depicts multiple, short synthetic oligomeric DNA sequences based on the sequences of the unique spacer DNA's within the DR region.
  • Figure 3 shows the genetic organisation of the structures of the DR-like loci in various bacterial species. * • depicts the transcription direction of open reading frame (ORF)
  • MTCY 16B7.26, 27 and 30C are unknown genes/proteins.
  • iap gene function is alkaline phosphatase isozyme conversion.
  • ORF f94, f305, YGCE and f223 are unknown genes/proteins.
  • ORFC and D are unknown genes/proteins and ORF 1A and IB are possibly transposases of IS elements 1000 and 1000A.
  • 0RF21 is an unknown gene/protein. Probably another repeat cluster is also present on the megaplasmid pHM500.
  • LR Long Repeat
  • thermoautrophicum Two repeat clusters SRI and SRII flanked by 21
  • LRI, LRU. LRI and LRU are almost identical and are homologues of the LR segment of M. jannaschii. SRI and SRII are separated by 500 kb in the genome .
  • CelA For Thermatoga maritima:CelA gene encodes cellulase: endo-l,4-beta- glucanase (EC 3-2.1.4) and CelB is also a cellulase exhibiting 58% identity with celA.
  • the SRIA and SRIB repeat clusters have the same Repeat Sequence and the SRII Repeat Sequence is also clearly homologous.
  • the SR clusters are separated by about 400bp.
  • SRIB and SRII are located near tRNA genes.
  • SRIA lies adjacent to an unknown 0RF3.
  • FIG. 4 Hybridization Patterns of 17 E. coli isolates. Thirty four different spacer oligonucleotides were covalently linked to a membrane and PCR amplified DNA of E. coli was hybridized as described (Kamerbeek et al. 1997) , except that the primers used to amplify the DR locus were specific for the DR sequence from E. coli. Note the polymorphism observed in E. coli due to the strain-dependent presence or absence of spacer DNA.

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Abstract

Dans le domaine de la différentiation de souches de bactéries entre elles, un procédé général a été mis au point selon lequel divers types de bactéries peuvent être différentiés en souches individuelles séparées. Par conséquent, ce procédé peut être utilisé dans le milieu clinique pour déterminer la souche de bactéries qui est présente dans un échantillon. Ce nouveau procédé peut être appliqué pour discerner différentes souches de bactéries de type tant Gram positif que Gram négatif.
EP98912806A 1998-04-03 1998-04-03 Procede de differentiation de souches de bacteries Withdrawn EP1066407A1 (fr)

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US7349808B1 (en) * 2000-09-06 2008-03-25 Egenomics, Inc. System and method for tracking and controlling infections
EP1409738A4 (fr) 2001-06-22 2005-03-30 Marshfield Clinic Procedes et oligonucleotides pour la detection de salmonella sp., e. coli o157:h7, et de listeria monocytogenes
EP1828422A4 (fr) * 2004-11-15 2008-07-30 Bacterial Barcodes Inc Detection au moyen d'amorces vers l'adn repetitif et amplification basee sur la transcription par une telle detection
US10066233B2 (en) 2005-08-26 2018-09-04 Dupont Nutrition Biosciences Aps Method of modulating cell resistance
US7531309B2 (en) 2005-12-06 2009-05-12 Michigan State University PCR based capsular typing method
FR2925918A1 (fr) * 2007-12-28 2009-07-03 Pasteur Institut Typage et sous-typage moleculaire de salmonella par identification des sequences nucleotidiques variables des loci crispr
US20220389484A1 (en) * 2019-06-06 2022-12-08 Tata Consultancy Services Limited System and method for combating infections due to pathogens belonging to phylum proteobacteria

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WO1993008297A1 (fr) * 1991-10-23 1993-04-29 Baylor College Of Medicine Determination d'empreintes relatives a des souches bacteriennes par amplification repetitive de sequences d'adn
US6074820A (en) * 1994-05-16 2000-06-13 De Staat Der Nederlanden, Vertegenwoordigd Door De Minister Van Welzijn, Volksgezondhed En Cultuur Detection and differentiation of mycobacterium tuberculosis complex bacteria by direct variant repeat oligotyping

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