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WO1997001647A2 - Sequences d'adn pour identifier des lignees hautement transmissibles de pseudomonas (burkholderia) cepacia - Google Patents

Sequences d'adn pour identifier des lignees hautement transmissibles de pseudomonas (burkholderia) cepacia Download PDF

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Publication number
WO1997001647A2
WO1997001647A2 PCT/US1996/011132 US9611132W WO9701647A2 WO 1997001647 A2 WO1997001647 A2 WO 1997001647A2 US 9611132 W US9611132 W US 9611132W WO 9701647 A2 WO9701647 A2 WO 9701647A2
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sense primer
variant
dna
cepacia
pseudomonas cepacia
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WO1997001647A3 (fr
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Richard Goldstein
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Trustees Of Health & Hospitals Of City Of Boston Inc
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Trustees Of Health & Hospitals Of City Of Boston Inc
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Priority to AU64041/96A priority Critical patent/AU6404196A/en
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Publication of WO1997001647A3 publication Critical patent/WO1997001647A3/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/21—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Pseudomonadaceae (F)
    • 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

  • This invention relates generally to the detection of highly transmissible strains of Pseudomonas (Burkholderia) cepacia, * and particularly to the use of DNA based fingerprints and sequences for identifying such epidemic lineages.
  • Pseudomonas cepacia is an aerobic, gram- negative bacillus with an ubiquitous distribution in soil and water. In recent years this organism has emerged as an important pathogen among cystic fibrosis (CF) patients. CF patients with respiratory colonization or infection with P. cepacia have higher morbidity and mortality than those CF patients not infected by this organism. While the significant increase in P. cepacia infection suggests epidemic spread ' , the source and transmissibility of
  • P. cepacia remains controversial . Nonetheless, given the potentially grave consequence of P. cepacia infection, stringent infection control policies have been adopted, many CF camps in North America have been closed and all but one lung transplant center have ceased to accept P. cepacia-infected CF patients as transplant candidates.
  • RFLP chromosomal restriction fragment length polymorphism
  • the present invention provides DNA based fingerprints for identifying highly transmissible lineages of Pseudomonas cepacia produced by: (a) ribotyping analyses, i.e., the determination of RFLPs associated with the multicopy RNA operon(rrn); and (b) pulsed field gel electrophoresis (PFGE)-based resolution of chromosomal macro-RFLP patterns.
  • Another object of this invention is to provide isolated DNA molecules encoding a 17-KDa major subunit pilin protein (cblA) of cystic fibrosis- associated, epidemic and non-epidemic Pseudomonas cepacia strains.
  • the recombinant polypeptides are then used to produce antibodies for use in methods for identifying epidemic strains of Pseudomonas cepacia .
  • PCR polymerase chain reaction
  • Figs 1 A-C together depict RFLPs of 17 P. cepacia isolates cited in the specification. Lane order for the 17 isolates is maintained in all three figures. Isolate numbers of examined strains appear at the top of the figure immediately above each lane. Subscript letters preceding isolate number indicate CF center from which P. cepacia (PC) strain was isolated: PC E , Edinburgh Scotland; PC MS , Jackson MS; PC N , Chapel Hill NC; PC NY , New York NY; PC 0H , Cleveland OH; PC T , Toronto Canada, and C V / Norfolk VA.
  • PC P. cepacia
  • Fig. IA. PFGE-resolved Spel RFLPs.
  • samples were prepared and restriction fragments separated by pulsed field gel electrophoresis with a CHEF Mapper system (BIO-RAD) through 1% agarose using a field strength of 6 V/cm and an initial and final pulse time of 1.2 sec and 54 sec, respectively. Fragment sizes were determined using a ⁇ concatenate ladder. Bar-code format translation of chromosomal fingerprint profiles was made using a
  • Fig. IB rrn EcoRI RFLPs. Southern blot hybridization methods were as we described
  • Fig. 2 rrn-RFLP based phylogenic tree of representative isolates from patients at seven CF centers in North America (Chapel Hill NC, Jackson MS, Norfolk VA, Cleveland OH, Philadelphia PA, New York NY, Toronto Ontario) and Europe (Edinburgh) plus environmental and clinical (non-CF) sources. All cited isolates are described in the text and in the Methods section. Indicated isolate number is followed by
  • cblA ⁇ isolate (s) that encode the cblA gene (Fig. IC) and express adhesin Cbl pili (Fig. 3) .
  • cblA-t_ identical 501 bp sequence carried by Toronto and Edinburgh CF center isolates (Fig. 4) ;
  • cblA 2 polymorphic 501 bp sequence carried by Jackson
  • Fig. 3 Transmission electron micrograph of Toronto epidemic strain PC T _7 expressing Cbl adhesin pili. High resolution was achieved with a JOEL 100 CX electron microscope as pre- 11 electron microscope as previously described . Bar in lower right corner, 0.1 ⁇ m
  • Fig. 4 Identical 501 bp sequence (top-most line) of the cblA structural gene encoded by 2 prototypic Toronto epidemic isolates (PC T _7 and PC T _5) and 2 prototypic Edinburgh isolates (PC E _2315 and PC E _ 1359) compared to the variant cblA sequence carried by the single Jackson Mississippi CF center isolate PC ⁇ g. 2323 (lower line) .
  • One aspect of the present invention is directed to the identification of highly transmissible strains of Pseudomonas cepacia using DNA based fingerprints. More specifically, such identification
  • SUBST ESHEET(RUL!2S is accomplished by the use of fingerprints produced by: (a) ribotyping analyses, i.e., the determination of restriction fragment length polymorphisms (RFLPs) associated with the multicopy RNA operon (rrn) ; and (b) pulsed field gel electrophoresis (PFGE)-based resolution of chromosomal macro-RFLP patterns.
  • ribotyping analyses i.e., the determination of restriction fragment length polymorphisms (RFLPs) associated with the multicopy RNA operon (rrn)
  • PFGE pulsed field gel electrophoresis
  • DNA samples from bacterial strains are isolated, digested with a restriction endonuclease such as EcoRI and separated by agarose gel electrophoresis.
  • the DNA fragments from the agarose gel are then transferred to nitrocellulose membranes and probed with either radiolabeled or chemiluminescent E. coli ribosomal RNA ⁇ rRNA) probes.
  • the RFLPs detected by hybridization with the E. coli rRNA probes are then analysed to categorize the bacterial strains according to their distinctive bands of rRNA encoding DNA (i.e., M DNA fingerprints").
  • isolated DNA molecules encoding a 17-KDa major subunit pilin protein (cblA) of cystic fibrosis-associated, epidemic and non-epidemic Pseudomonas cepacia strains are provided.
  • the genes encoding the cblA protein are isolated and sequenced by standard techniques.
  • the cblA genes isolated from epidemic and non-epidemic strains of Pseudomonas cepacia enabled for the first time the comparison of those variant genes and the identification of the differences in their nucleotide sequences.
  • the isolated DNA molecules are then used for the recombinant production of the 17-kDa major subunit pilin protein (cblA) of cystic fibrosis-associated, epidemic and non-epidemic Pseudomonas cepacia strains.
  • cblA major subunit pilin protein
  • the present invention also contemplates recombinant DNA molecules containing the above DNA molecules and unicellular hosts transformed with those recombinant DNA molecules.
  • the recombinant polypeptides and their fragments are then used for the production of antibodies that can distinguish epidemic and non-epidemic strains of Pseudomonas cepacia in
  • SUBSTTTi ⁇ i SHEET (RULE 2 ⁇ ) standard immunologic assays such as ELISA, radioimmunoassay and western blots.
  • ELISA enzyme-linked immunosorbent assay
  • radioimmunoassay enzyme-mediated immunologic assay
  • western blots standard immunologic assays
  • the methods for recombinant protein production, protein purification and generation of antibodies are well within the purview of the ordinary skilled artisan.
  • This invention also provides unique primer oligonucleotide sequences derived from cblA gene variants for use in polymerase chain reaction (PCR)- based methods for detection of highly transmissible strains of Pseudomonas cepacia .
  • PCR polymerase chain reaction
  • These unique primers are usually synthesized using standard procedures following identification of desirable nucleotide sequences based on the comparison of the cblA gene sequences of epidemic and non-epidemic strains of Pseudomonas cepacia .
  • the PCR techniques contemplated by and used in the present invention are well known. Essentially, the primers, DNA from the bacterial strain to be tested and a thermostable PCR enzyme are mixed and the reaction carried out according to established procedure in a thermocycler block. The PCR products are then analyzed generally by electrophoretic separation.
  • the invention also provides DNA probes derived from unique regions of variant cblA gene sequences that may be used in standard hybridization based assays such as colony hybridization or Southern blot transfers to detect highly transmissible strains of Pseudomonas cepacia .
  • the present invention also contemplates the use of the above DNA based fingerprints, oligonucleotide primers, DNA hybridization probes, polypeptides and antibodies in diagnostic kits for the
  • 133 Pseudomonas cepacia isolates were obtained from the following sources: (i) sixty-five isolates from patients at the University of North Carolina Cystic Fibrosis Center (1985 through 1993) including 17 clinic and 5 transplant patients, 4 of whom were infected transfers from other distant sources: (i) sixty-five isolates from patients at the University of North Carolina Cystic Fibrosis Center (1985 through 1993) including 17 clinic and 5 transplant patients, 4 of whom were infected transfers from other distant
  • Chromosomal DNA was prepared using the following procedure. Overnight cultures were diluted 10-fold in 10 ml of LB and incubated at 37°C until they reached mid-log phase. Cells were pelleted, washed twice with 0.9% NaCL, and then resuspended in cold TE (10 mM Tris-HCl, 10 mM EDTA [pH 8.0]). Lysozyme was added to a final concentrations of 20 mg/ml, and the solution was incubated at 37°C for 30 min. Proteinase K (20 mg/ml in TE; Sigma) and sodium dodecyl sulfate were added to final concentration of 20 ⁇ g/ml and 0.1%, respectively. The lysates were incubated overnight at 50°C. Sarcosine-free acid (Sigma) was added to a final concentration of 2%, and the solution was mixed gently.
  • the DNA was then purified by cesium chloride-ethidium
  • the DNA restriction fragment used to generate the probe was separated by horizontal slab gel electrophoresis in 0.8% low-melting point agarose.
  • the relevant restriction fragment from a slice of the gel was radiolabeled in the agarose by random
  • the chromosomal DNA prepared according to the above-described method was restricted with JEcoRl and the fragments were separated by agarose gel electrophoresis. Following electrophoresis, restriction fragments were transferred to nitrocellulose membranes and hybridized with the rrnB probe described above. Hybridized bands were visualized by autoradiography and the banding patterns
  • Pulsed-field gel electrophoresis Cells were grown to early log phase in LB, harvested by centrifugation, washed with 1:1 TE buffer, resuspended in 1 ml of 10:1 TE buffer, and mixed with 1.2 volumes of melted 1% InCert agarose (Bio-Rad) in TE buffer. The mixture was dispensed into 120- ⁇ l insert molds (Pharmacia) and allowed to solidify on ice. Plugs were sliced and incubated in 20 ⁇ g of lysozyme per ml at 37°C for 12 h.
  • the lysozyme buffer was replaced with ESP buffer (0.5 MEDTA [pH9], 1% sarcosyl, 200 ⁇ g of proteinase K [Sigma] per ml) , and the plugs were incubated at 37°C for 5 h and then washed with TE buffer for 4 h at 37°C. Single plug slices were incubated with Spel (Boehringer Mannheim) in restriction enzyme buffer for 2 h.
  • ESP buffer 0.5 MEDTA [pH9], 1% sarcosyl, 200 ⁇ g of proteinase K [Sigma] per ml
  • strains were assigned to the same ribotype when comparison of sizes of hybridizing fragments revealed 3 or fewer bands differing between the two patterns under comparison.
  • PFGE chromosomal fingerprints were considered different (i) when they had the same number of DNA fragments but when the size of at least one band varied by more than I standard deviation (5%), (ii) when they exhibited a different number of DNA fragments, or (iii) when the sum of the sizes of the differing bands in the first PFGE pattern did not correspond to that of the differing DNA fragments in the second PFGE pattern.
  • D>0.90 represents closely related strains, while unrelated strains have D ⁇ 0.60. Intervening values, remarkably, were not observed, and values between 0.5
  • 21 cblA gene was PCR amplified using a DNA thermocycler
  • Method A This method was based upon three 'sets' of VARIANT EXTERNAL (downstream to 3' end of cblA gene) anti-sense PCR primers and COMMON INTERNAL sense primers (at 5' end of C- JA gene) . Each set specifically amplifies only one or another of the 3 known variant cblA gene sequences, including that uniquely associated with the Toronto/Edinburgh (T/E) epidemic clone. • To amplify only T/E* epidemic lineage clones the following primers were used to generate a 573 base pair (bp) PCR product: common sense primer:
  • T/E (Toronto/Edinburgh) the sough ⁇ after, highly transmissible, epidemically spread cblA clone, for example isolates PC T -5 clone, PC T -7, PC E -1359, PC E -2315.
  • ** 1-20 in accord with conserved region of cblA gene sequence, numbered as in Fig. 4.
  • MSp-type non-epidemic, negligible transmissibility cblA strain from Mississippi, for example isolate PC MS -2323.
  • ** 243-260 in accord with cblA gene sequence numbering in Fig. 4 for epidemic isolates PC T -5, PC T -7, PC E -1359, PC E -2315.
  • This method was based upon three 'sets' of Internal (within cbJA gene) PCR primers. Each set specifically amplifies only one or another of the 3 known variant cblA gene sequences, including that uniquely associated with the T/E epidemic clone.
  • METHOD Cl was based on 'common' sense primer and variant antisense primers.:
  • ** 1-20 in accord with conserved region of cblA gene sequence, numbered as in Fig. 4.
  • this (MS w -type) is a recently discovered variant cblA gene sequence carried by another non- epidemic strain of negligible transmissibility from Mississippi.
  • the probability of the random occurrence of 16- to 19-base long sequences, such as the specific primers employed in any of the above methods, is once every 4 repeatedly 16 /(or ca. one i•n 1 ⁇ r0s 9 s) 4t-o 4/.19 /(or ca. one in 10 11) bp x 2. Given that the size of the P. cepacia genome is ca. 7 x 10 bp, the chance occurrence would be remote.
  • any one of the above pairs of primers corresponding to the T/E epidemic lineage is used in standard PCR reactions with DNA from the bacterial strain to be tested.
  • reaction products are analyzed for the presence of the appropriate size PCR product using standard methods.
  • Method A when the pair of primers for the T/E epidemic lineage is used, the detection of a 573 bp PCR product by electrophoresis on an agarose gel would indicate that the sample contains a strain of the highly transmissible T/E. Whereas, the absence of such a PCR product would indicate otherwise.
  • Methods B, Cl and C2 the presence of a strain of the highly transmissible T/E would be confirmed by PCR products of 331 bp, 427 bp and 185 bp, respectively.
  • Hybridization-specific detection based on 'probes' derived from unique regions of variant cblA gene sequences:
  • Each unique probe sequence shown below can be generated by restriction endonuclease double digestion of one or another of the three cblA gene variants, based on:
  • the probes for the T/E lineage disclosed above can be used in standard hybridization methods, such as colony hybridization assays or Southern blot transfer, to detect the presence of the highly transmissible P. cepacia strain of the T/E lineage.
  • Edinburgh isolates comprise a single, clonally-related lineage, only distantly related to all other isolates, and (ii) the remaining, independently isolated strains from other CF centers are as distantly-related to one another as they are either to the Toronto/Edinburgh clusters or to the independently isolated non-CF clinical and environmental strains. We then examined the epidemic Edinburgh
  • Genotypic survey was carried out by stripping rrn-probe from an EcoRI chromosomal digest membrane (Fig. IB) followed by hybridization with a cb-ZA probe.
  • Fig. IC (lanes 1-8) indicates that four highly transmissible Edinburgh isolates as well as the closely-related Toronto clones encode cblA.
  • Burkholderia (Pseudomonas) cepacia. J. Bacteriol. 177, 1039-1052 (1995) .

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Abstract

L'invention concerne des empreintes et des séquences d'ADN permettant d'identifier des lignées hautement transmissibles de Pseudomonas cepacia. Plus particulièrement, l'invention concerne des motifs de bandes génétiques ou des empreintes d'ADN permettant d'identifier des lignées hautement transmissibles de Pseudomonas cepacia. Ces motifs sont obtenus (a) par analyse des ribotypes, c'est-à-dire la détermination du polymorphisme de fragments de restriction (RFLP) associés à l'opéron d'ARN en copies multiples et (b) par électrophorèse sur gel à champs pulsés pour la résolution de motifs macro-RFLP chromosomiques. L'invention concerne également des séquences spéciales d'amorces oligonucléotidiques et de sondes d'ADN dérivées des variants d'un gène, codant une sous-unité majeure de 17 KDa de la protéine piline (cb1A) de Pseudomonas cepacia associé à la mucoviscidose, pour une utilisation dans des procédés PCR pour la détection de souches hautement transmissibles de Pseudomonas cepacia. L'invention concerne également des procédés et des trousses permettant de diagnostiquer des lignées hautement transmissibles de Pseudomonas cepacia, qui utilisent les empreintes d'ADN, les amorces oligonucléotidiques et les sondes d'ADN en question. Un autre objet de cette invention est de fournir de molécules d'ADN isolées codant une sous-unité principale de 17 KDa de la protéine piline de certaines souches de Pseudomonas cepacia associées à la mucoviscidose, des molécules d'ADN de recombinaison, des hôtes transformés et des procédés de production de cette protéine. L'invention concerne également des anticorps contre la sous-unité majeure de 17 Kda de la protéine piline.
PCT/US1996/011132 1995-06-28 1996-06-28 Sequences d'adn pour identifier des lignees hautement transmissibles de pseudomonas (burkholderia) cepacia Ceased WO1997001647A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000014274A1 (fr) * 1998-09-03 2000-03-16 The University Of British Columbia PROCEDE D'IDENTIFICATION ET DE SPECIATION DE BACTERIES DU COMPLEXE $i(BURKHOLDERIA CEPACIA)
US6630302B1 (en) * 1997-07-25 2003-10-07 The Trustees Of Boston University Methods and compositions for determining species of bacteria and fungi
RU2458140C1 (ru) * 2011-02-17 2012-08-10 Федеральное государственное учреждение здравоохранения "Волгоградский научно-исследовательский противочумный институт Федеральной службы по надзору в сфере защиты прав потребителей и благополучия человека Способ выявления экспрессирующихся генов патогенных буркхольдерий методом дифференциального дисплея

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Title
CLINICAL MICROBIOLOGY REVIEWS, vol. 7, no. 3, 1 July 1994, pages 311-327, XP000604155 BINGEN E H ET AL: "USE OF RIBOTYPING IN EPIDEMIOLOGICAL SURVEILLANCE OF NOSOCOMIAL OUTBREAKS" *
INTERNATIONAL CONGRESS SERIES, 21 May 1993, pages 95-99, XP000604136 RYLEY H C ET AL: "TYPING OF PSEUDOMONAS CEPACIA ISOLATES FROM WELSH CF PATIENTS BY PCR OF RNA RIBOSOMAL GENES" *
JOURNAL OF BACTERIOLOGY, vol. 177, no. 4, 1 February 1995, pages 1039-1052, XP000604151 GOLDSTEIN R ET AL: "STRUCTURALLY VARIANT CLASSES OF PILUS APPENDAGE FIBERS COEXPRESSED FROM BURKHOLDERIA (PSEUDOMONAS) CEPACIA" *
JOURNAL OF BACTERIOLOGY, vol. 177, no. 4, February 1995, pages 1030-1038, XP000603985 SAJJAN U S ET AL: "CABLE (CBL) TYPE II PILI OF CYSTIC FIBROSIS-ASSOCIATED BURKHOLDERIA (PSEUDOMONAS) CEPACIA: NUCLEOTIDE SEQUENCE OF THE CBLA MAJOR SUBUNIT PILIN GENE AND NOVEL MORPHOLOGY OF THE ASSEMBLED APPENDAGE FIBERS" *
JOURNAL OF CLINICAL MICROBIOLOGY, vol. 32, no. 10, October 1994, pages 2422-2424, XP000604176 DASEN S E ET AL: "CHARACTERIZATION OF PCR-RIBOTYPING FOR BURKHOLDERIA (PSEUDOMONAS) CEPACIA" *
JOURNAL OF CLINICAL PATHOLOGY, vol. 47, no. 3, March 1994, pages 222-226, XP000603979 O'CALLAGHAN E M ET AL: "DEVELOPMENT OF A PCR PROBE TEST FOR IDENTIFYING PSEUDOMONAS AERUGINOSA AND PSEUDOMONAS (BURKHOLDERIA) CEPACIA" *
NATURE MEDICINE, vol. 1, no. 7, July 1995, pages 661-666, XP000605148 SUN L ET AL: "THE EMERGENCE OF A HIGHLY TRANSMISSIBLE LINEAGE OF CBL PSEUDOMONAS (BURKHOLDERIA) CEPACIA CAUSING CF CENTRE EPIDEMICS IN NORTH AMERICA AND BRITAIN" *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6630302B1 (en) * 1997-07-25 2003-10-07 The Trustees Of Boston University Methods and compositions for determining species of bacteria and fungi
WO2000014274A1 (fr) * 1998-09-03 2000-03-16 The University Of British Columbia PROCEDE D'IDENTIFICATION ET DE SPECIATION DE BACTERIES DU COMPLEXE $i(BURKHOLDERIA CEPACIA)
GB2362212A (en) * 1998-09-03 2001-11-14 Univ British Columbia Method for the identification and speciation of bacteria of the burkholderia cepacia complex
RU2458140C1 (ru) * 2011-02-17 2012-08-10 Федеральное государственное учреждение здравоохранения "Волгоградский научно-исследовательский противочумный институт Федеральной службы по надзору в сфере защиты прав потребителей и благополучия человека Способ выявления экспрессирующихся генов патогенных буркхольдерий методом дифференциального дисплея

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