WO1996019585A1 - Typage de micro-organismes - Google Patents

Typage de micro-organismes Download PDF

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WO1996019585A1
WO1996019585A1 PCT/AU1994/000781 AU9400781W WO9619585A1 WO 1996019585 A1 WO1996019585 A1 WO 1996019585A1 AU 9400781 W AU9400781 W AU 9400781W WO 9619585 A1 WO9619585 A1 WO 9619585A1
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region
strains
rrna
primers
amplification
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PCT/AU1994/000781
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English (en)
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Volker GÜRTLER
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Heidelberg Repatriation Hospital
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Priority to PCT/AU1994/000781 priority Critical patent/WO1996019585A1/fr
Priority to AU13075/95A priority patent/AU1307595A/en
Publication of WO1996019585A1 publication Critical patent/WO1996019585A1/fr

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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
    • C12Q1/6876—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 to a method of detecting, identifying and quantitating microorganisms, and to oligonucleotide probes for use in this method.
  • the method relates to the typing of specific isolates of microorganisms, and discrimination between strains and allelic subtypes.
  • immunological identification which usually requires the use of monoclonal antibodies. A prerequisite for such immunological identification is that the species of the organism in question be known, or at least strongly
  • Other typing methods which can be used for certain species only, include toxin detection, isolation of plasmids, bacteriophage, bacteriophage/bacteriocin typing systems, antibiotic susceptibility testing, protein typing by SDS-polyacrylamide gene electrophoresis, pulsed-field gel electrophoresis, immunoblotting, and restriction endonuclease analysis.
  • rrn The rRNA operon, rrn, is present in varying copy number in all bacteria, with some regions highly conserved and others highly variable (Neefs et al, 1990).
  • rRNA operon has a very high genetic stability and the length of the 16S rRNA gene is constant in all eubacteria (Neefs et al, 1990), the number of rRNA operons has been completely analyzed by Southern
  • the probes are species-specific, and are
  • oligonucleotide probe is required for each microorganism species.
  • U.S. Patent No. 5,288,611 by Kohne describes methods and probes for identification and quantification of any organism or group of organisms containing rRNA, including previously unknown organisms. Probes specific for individual species and for groups of related species, and Probes hybridizing to rRNA or to tRNA are described.
  • U.S. Patent No. 5,292,874 by Milliman discloses hybridization probes specific for Stapbylococcus aureus probes, which detect a unique rRNA sequence in the 23S rRNA gene.
  • Japanese Patent Publication No. 6090793 by Takara Shuzo Co. Ltd. describes methods for detection of bacteria of the genus Lactobacillus, by detection of a sequence in the spacer region between the gene encoding 16S rRNA and the gene encoding 23S rRNA.
  • No. 6090793 requires a separate oligonucleotide for each species of organism. None of these specifications mentions the existence or number of rrn alleles, or describes a method permitting differentiation of strains within a species, or of allelic variations.
  • PCR analysis of the 16S rRNA gene has been used to demonstrate species-specific differences (Gürtler et al, 1991) and strain differences (Vaneechoutte et al, 1992) in various bacterial species. Allelic species-specific differences within the 16S rRNA gene have been demonstrated in clostridia (Gürtler et al, 1991). The rRNA alleles of E. coli (Brosius et al, 1981) and B. subtilis (Loughney et al, 1982) have been shown to have variable length
  • oligonucleotide primers (A, B, C, D, E) used in the prior art and in the present invention.
  • the abbreviations ile and ala refer to the respective genes-encoding tRNA for isoleucine and alanine.
  • Primers A and E are as disclosed in WO 93/11264.
  • Figure 2 illustrates the approaches used for the detection of rRNA alleles in C. difficile by Southern hybridization and PCR.
  • the hatched bars (A, B, C and D) show positions of the respective PCR products (Table 3), the shaded bar denotes the 16S rRNA gene, the solid bar denotes the 23S rRNA gene, and the line joining the 16S and 23S gene depicts the spacer regions.
  • the HindiII site is at position 975 of the 16S rRNA gene (Gürtler et al, 1991).
  • Figure 3 shows hybridization of PCR product B to Group II bands in genomic DNA isolated from C. difficile and C. bifermentans strains.
  • Lane 14 pBR328 DNA digested with BgII and HinfI, labelled with photodigoxigenin;
  • Y indicates the position of an extra band visible in C. bifermentans products.
  • Figure 4 illustrates the hybridization of PCR product A to Group I and II bands in genomic DNA isolated from C. difficile strains.
  • Lane 1 pBR328 DNA digested with BgII and HinfI, labelled with photodigoxigenin;
  • Figure 5 shows the detection of rRNA alleles in C. difficile strains by Southern hybridization. The symbols, box shadings and the position of the HindIII site are described in the legend to Figure 2. Bands depicted as Group I ( Figures 3 and 4) correspond to fragments 5' of the HindiII site and Group II bands ( Figures 4 and 5)
  • ⁇ refers to bands which are not present in all strains.
  • Figure 6 shows the constant and variable length regions within PCR product C amplified from C. difficile strains, as demonstrated by agarose gel electrophoresis of undigested (lanes 2-7) and HindIII-digested (lanes 10-15) PCR product C.
  • Lane 1 pBR328 DNA digest with HinfI and BglI ;
  • Lane 9 pBR328 DNA digested with HinfI and BglI ; Lanes 10-15, h13, H14, H17, H19, H23 and 630, respectively.
  • the standards are 2176, 1766, 1230, 1033, 653, 517, 453, 394, 298, 234 and 220 bp respectively.
  • V d variable HindIII-digested.
  • Figure 7 shows denaturing PAGE of PCR product C amplified from strains of C. difficile.
  • the sizes of the respective alleles are shown on the left [mean ⁇ SEM (number of determinations)].
  • the molecular mass markers used (not shown) were ⁇ DNA digested with HindIII and EcoRI (947 and 831 bp bands only) and SPPI DNA digested with EcoRI (1150 and 1000 bp bands only).
  • Figure 8 is a dendrogram showing the
  • C. difficile ribotypes Using maximum parsimony, 50 equally parsimonious trees were found, one of which is shown. The same ribotypes were found in each of the circled branches (a, b, c) for all 50 trees. The root of the tree (C. bifermentans) had no bands in common with any of the ribotypes.
  • Figure 9 shows the distribution of rRNA genes and restriction sites in the region of interest in
  • Staphylococcus aureus The solid line joining these genes can vary in length in the same strain or in different strains ( Figure 12b).
  • the dashed lines show positions of the PCR products C, I & J, which were obtained using the primers R1392F and LR488, SP1F and SP2R and SP1F and LR20F respectively (Table 8).
  • the dotted lines show the origins of HpaII fragments (E, F, G and H) obtained from PCR product C. The locations of other primer binding regions that were used to sequence HpaII fragment E are also shown.
  • Figure 10 illustrates denaturing PAGE of PCR products amplified from strains of S. aureus.
  • MRSA Methicillin resistant S. aureus
  • Lanes 1-6 Ribotypes Pi, Pj, PF A(strain H11);
  • Lanes 9-16 B, Mi, Mh, Mi, Pi, Mh and Mj.
  • the sizes of the respective alleles are shown on the right [mean ⁇ SEM (number of determinations)].
  • the molecular mass markers (lanes 7 & 8) used were ⁇ DNA digested with HindIII and EcoRI (1375 and 947 bp bands only) and SPP1 DNA digested with EcoRI (1150 and 1000 bp respectively).
  • Figure 11 shows the alignments of 16S-23S spacer sequences from S. aureus.
  • PCR product C from S. aureus strains (Table 9) was cloned into M12mp18RF and sequenced with the primers listed in Table 8 and Figure 9. The sequences were derived from the clones and isolates listed in Table 7.
  • the sequences SA16S and SA223S were taken from Ludwig et al (1992).
  • the alignment of rrn alleles with SA16S (a), rrnC, E, F, G, H, J, K & L (b) and rrn alleles SA23S (c) is shown.
  • Figure 12 is a dendrogram showing the
  • the method of the invention avoids cumbersome steps required by previously available methods, and is suitable for testing large numbers of samples; it is also amenable to automation.
  • the method of the invention is particularly suitable for epidemiological studies, for example
  • the invention According to a first aspect, the invention
  • a method of identification of microorganisms comprising the steps of extracting and purifying DNA from a sample suspected to contain bacteria, and subjecting the 16S-23S rRNA spacer region of said DNA to amplification, using a first primer comprising a sequence from the 5' end of the 16S rRNA gene, and a second primer comprising a sequence from the 3' end of the 23S rRNA gene, thereby producing fragments having detectable differences in size and number, and separating the amplified fragments.
  • the amplified fragments produced in the method of the invention are of variable length, they can be analysed directly, for example by electrophoresis; no other experimental step, such as hybridzation, is necessaryy in order to demonstrate differences between strains, although in some situations a hybridization step could be
  • the amplified products may be separated by any method which provides sufficient resolution.
  • other separation methods such as capillary electrophoresis or high performance liquid chromatography, may be used.
  • additional probes may be used, for example comprising the sequence encoding tRNA ile and/or the sequence encoding tRNA ala .
  • the sample will usually be a clinical sample such as blood, tissue, urine, faeces, sputum etc., a food sample, or an environmental sample such as a water sample or a soil sample. Other types of samples may be used, depending on the circumstances.
  • the DNA may be extracted by any suitable method, but preferably the method is a rapid one. Extraction with guanidine hydrochloride or by boiling water followed by column purification are both suitable. In some cases, particularly where clinical specimens are used, it may be advantageous to effect a preliminary purification of the sample following DNA extraction. If the nature of the bacteria sought to be tested is known, this may be carried out using monoclonal antibody methods, such as those using monoclonal antibody conjugated to magnetic beads . Some broad spectrum antibodies are also available for this purpose.
  • the primers used correspond to a highly conserved region from the 3' end of the spacer region, and to a highly conserved region from the 5' end of the spacer region respectively.
  • the invention provides amplification primers for use in the method of the invention. As described above, these primers correspond to highly conserved regions from the 3' end of the
  • 16S-23S rRNA spacer region respectively.
  • they correspond to regions from the 5' end of the 16S rRNA gene and to a region from the 3' end of the 23S rRNA gene respectively.
  • the primers are 15 to 20
  • primers are R1391F and LR488 or LR194F as herein defined. Most preferably LR488 is 15 to 19 nucleotides long, and R1391F is 15 to 18 nucleotides long. Primer C (LR488) is particularly preferred, because it is more highly conserved than primer A.
  • Clostridium difficile The identity of all strains of Clostridium difficile was determined by biochemical tests (Cato et al, 1986) and confirmed by gas-liquid chromatography (Sutter et al, 1985). Purified stocks were stored in cooked-meat broth at room temperature or in glycerol broth at -20°C. All strains were grown in brain heart infusion broth (BHI, Gibco). The stability of ribotype patterns was tested by passaging single colonies from horse blood agar plates every 2-3 days over a 5 week period. Toxin B production by C. difficile strains was detected by the method of
  • Genomic DNA and plasmid DNA was isolated by the protocol of Gürtler et al (1991), except that the cell walls of S. aureus were disrupted by incubating the strain with 200 g lysostaphin ml -1 at 37°C for 5-10 min.
  • DNA regions were amplified by the protocol of Gurtler (1991), except that the reaction volume and amount of DNA were halved, and 1.25 units Taq polymerase
  • PCR products R907-LR507 and R1391-LR507 were digested singly or doubly with 10-15 units HindiII and CfoI , as instructed by the manufacturer (Boehringer). Genomic DNA was digested with 30 units HindIII. The digested and undigested PCR products were resolved on 2% (w/v) low-gelling-temperature plus 2% (w/v) 'AR' agarose gels. The HindIII-digested genomic DNA was resolved on 1% (w/v) 'AR' agaorse gels.
  • PCR product M13F-M13R was digested with 10-15 units Dral or HinfI , as instructed by the manufacturer (Boehringer). Genomic DNA was digested with 20 units HpaII. The digested PCR products and genomic DNA were resolved on 2% w/v low-gelling-temperature plus
  • Sequencing was performed by the dideoxynucleotide method of Sanger et al (1977) using the Bst DNA sequencing kit (BioRad). 7-Deaza-2'-deoxyguanosine triphosphate was used to minimize band compression due to GC-rich regions.
  • DNA sequences were processed and analysed by the following methods.
  • the DNASIS program version 6;
  • FIG. 2 and Table 4 Products A and B were hybridized to HindIII-digested genomic DNA isolated from C. difficile and C. bifermentans strains. Differences in HindIII sites on both flanking sides of the 16S rRNA gene were sought within and between strains. Products C and D were amplified from C. difficile strains in an attempt to find differences in the length of the 16S-23S spacer region within and between strains.
  • the positions of all the primers are in regions which are highly conserved in eubacteria (Neefs et al, 1990; Guttell & Fox, 1988).
  • the nucleotide numbering system is that of E. coli operon (Brosius et al, 1978).
  • the positions of each product are schematically represented in Figure 2.
  • Products A and B consist only of parts of the 16S rRNA gene (Table 4).
  • the 16S rRNA gene is of constant length between alleles and strains of C. difficile (Gürtler et al, 1991).
  • Figure 3 When PCR product B was hybridized to C. difficile genomic DNA, Group II bands hybridized predominantly ( Figure 3), the Group I bands hybridized faintly, because product B included 62 bp 5' of the HindIII site (1/10 of product B).
  • Figure 3 genomic DNA digested with HindIII
  • the Group II bands consist of the spacer region and part of the 23S rRNA gene. These Group II bands were of variable length, which could be explained by the presence of either a variable HindIII site or of an insertion within the spacer or the beginning of the 23S rRNA gene.
  • we amplified PCR products C and D both of which include the spacer regions (Table 4). When the product C primer combinations of Table 4 were used, several bands (V u ) of varying molecular masses were obtained from each C. difficile strain, as shown in
  • PCR product C was amplified from various C.
  • Table 2 were analysed, 24 strains were divided into 14 ribotypes, which are also shown in Table 5.
  • the dendrogram depicted in Figure 8 shows that 3 clusters (a, b, c) are found in all trees analysed.
  • ribotype G 2 isolates were cultured from one patient at different times; within ribotype E, 3 isolates were cultured from one patient at different times. All other isolates which had identical patterns (ribotypes D, E, F, G and H) were from different patients.
  • Product C was amplified from various C. difficile strains and separated by denaturing PAGE. Accumulated values (mean ⁇ SEM) taken from five separate electrophoresis runs are shown. The data include runs (PCR, DNA
  • novel molecular typing method of the invention may be applied to epidemiological studies of C. difficile. Since 500 bp of the 5' end of the 23S gene was amplified, it was possible that the observed heterogeneity of PCR products was due to an insertion within the first 500 bp of the 23S gene. This possibility is supported by several findings.
  • At least one extra cleavage site has been reported in the large rRNA subunit of Leptospira interrogans (Hsu et al, 1990) and Salmonella species (Hsu et al, 1992), producing several fragments smaller than 23S and a 90 bp intervening sequence has been shown to be excised during large subunit rRNA maturation (Burgin et al, 1990).
  • the results presented in this specification show that in C. difficile, 430 bp 3' from position 507 of the 23S rRNA gene was of constant length and the 16S-23S spacer DNA was of variable length between alleles.
  • E. coli (Fournier & Ozeki, 1985) contain tRNA genes which vary in length from 75-90 bp. Of the 7 rrn operons in E. coli, all contain from 1-3 tRNA genes
  • Genomic DNA was isolated from S. aureus as described above, and amplified using the primers described in Table 6 and Figure 9.
  • primers A and E are as disclosed in WO 93/11264.
  • PCR product C was amplified from the S. aureus strains listed in Table 3. These included 281 MRSA from four geographically distinct clinical sources and 48 penicillin or methicillin sensitive strains from a single Melbourne source; several methicillin-resistant or sensitive strains from type culture collections were also used. These strains yielded various amplified products, of which only the most intense bands were considered to be alleles. In total, 15 alleles, designated rrnA to rrnO, were
  • Ribotype A was the major ribotype found between 1960 and 1989 in Melbourne (19/22 strains), Singapore (7/9 strains), Ireland (9/9 strains). New York (1/1 strain) and UK (12/12 strains).
  • ribotype B was the major ribotype found at the Heidelberg Repatriation Hospital; 176 were ribotype B and 57 were ribotype A.
  • the sensitive strains showed considerably more variation in the presence or absence of bands, yielding an additional 26 ribotypes from the 48 strains studied.
  • Figure 10c shows some of these strains.
  • the MRSA ribotypes A, B and I included some of the penicillin or methicillin sensitive strains. The occurrence of the alleles in the various ribotype classes is summarized in Table 8.
  • PCR product C was amplified from various S.
  • ribotypes A, B, C, D and Pa The stability of ribotypes A, B, C, D and Pa was investigated by 30 serial passages of strains 9144, H11, H12, H14 and H21 over a six-week period. The ribotype was assessed after every fifth passage by visual comparison with reference patterns, and was found to be stable except for strains H12 and H21. Strain H12 was identified as ribotype B at all passages except the fifth, where rrnL appeared, making it ribotype A. Strain H21 was originally found to be ribotype D; however during the stability experiment it was found to be ribotype A at all passages subsequently investigated (the colonies from which the DNA prepared were used completely).
  • ribotype D (strain H21) could be explained by the loss of rrnH giving rise to the stable ribotype A.
  • the relative instability of ribotype B in the present study was due to the loss of rrnL. The frequency of such events is low
  • PCR product C from strains D46 (ribotype B), H11 and ATCC33952 (both ribotype A) was cleaved with HpaII and the resulting fragments cloned into M13 vectors.
  • Figure 1 was amplified as described above from S. aureus genomic DNA from strains D46, Hll, and 33952 using primers R1391F and LR488, R1391FH and LR488H or R1391F and LR194F
  • dithiothreitol 0.1mg bovine serum albumin ml -1 and
  • HpaII digested products (1-25ng) were ligated directly into AccI digested M13mp19RF (50ng: in a total of 10 ⁇ l) and the end repaired HpaII digested products (1-25ng) from strain D46 were ligated into Smal digested M13mp19RF (50ng: in a total of 10 ⁇ l) with 1 unit T4 DNA ligase (Boehringer), 66mM Tris-HCl (pH 7.5), 5mM MgCl 2 , ImM dithiothreitol,
  • M13 DNA was then prepared from all the positive clones (Sambrook et al, 1989). To determine the presence and size of the inserts, the single stranded DNA from the M13 clones was used as a template in the PCR using M13F and R primers which flank the SmaI and Accl
  • insert F With the sequence information of insert (H), primer LR194F was designed so as to contain a HpaII site. For strain 33952, primers LR194F and R1391F were used to obtain a mixture of PCR products, which were digested with Hpall to yield predominantly product E and cloned into Ml3mpl9; clones V18-V47 were isolated, and of these 9 contained insert E. These results are summarized in Table 9.
  • the 16S-23S rDNA spacer sequences of 9 rrn operons were determined from 3 methicillin resistant
  • the tRNA ile gene was present in rrnJ,G,F,C and A, while the tRNA ala gene was only present in rrnA and C.
  • the number of base pair differences between clones judged to be the same allele was 5 for rrnJ (from strains H11, D46 and 33952, isolated in 1982, 1992 and 1981 respectively), 2 for rrnH (from strains 33952 and H11), 4 for rmF (strain 33952) and 5 for rraC (33952).
  • the rrn alleles were divided into 3 distinct groups, which are shown in
  • tRNA gene found in the 16S-23S spacer varies in number (0, 1 or 2) and combination between operon and between species; A. bydropbila (East & Collins, 1993) and E. coli (Morgan et al, 1977) have tRNA ala , tRNA ile and tRNA glu ; B. subtilis (Loughney et al, 1982) and
  • Ribotype A was the major ribotype found (ribotype A/total no of strains in location) between 1960 and 1989 in Melbourne (19/22), Ireland (9/9), New York (1/1) and UK (12/12).
  • ribotype B was the major ribotype found at the HRH (176 were ribotype B and 57 were ribotype A).
  • the MRSA strains the MRSA strains
  • 16S-23S spacer makes it an ideal candidate for typing of strains and species identification which can potentially be applied to any species of the bacterial kingdom. Our method permits reliable, rapid identification and typing on this basis.
  • fragment C (31) can be obtained by adding 331bp (HpaII 2 to LR520) and 47bp (R1392 to HpaII 1 ) to fragment E ( ⁇ ).
  • Vaneechoutte M., Roussau, R., De Vos, P., Gillis, M., Janssens, D., Paepe, N., De Rouck, A., Fiers, T.,

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Abstract

La présente invention concerne un procédé permettant de détecter, d'identifier et de quantifier des micro-organismes, ainsi que les sondes oligonucléotidiques utilisées selon ce procédé. L'invention concerne particulièrement le typage d'isolats spécifiques de micro-organismes ainsi que la discrimination entre souches et sous-types alléliques. Selon le procédé présenté, on procède à l'amplification de la région d'espacement de l'ARN ribosomique 16S-23S au moyen d'une région extrêmement protégée provenant de l'extrémité 3' de la région d'espacement de l'ARN ribosomique 16S-23S, et/ou d'une région extrêmement protégée provenant de l'extrémité 5' de ladite région. Ce procédé rend possible un suivi épidémiologique des sous-types spécifiques de micro-organismes. L'invention privilégie certaines amorces et les revendiquent.
PCT/AU1994/000781 1994-12-20 1994-12-20 Typage de micro-organismes WO1996019585A1 (fr)

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DE102008002978A1 (de) 2008-06-09 2009-12-10 Universität Kassel Verfahren zur Differenzierung von verschiedenen Spezies der Gattung Methylobakterium
EP2163648A1 (fr) 2008-09-16 2010-03-17 Österreichische Agentur für Gesundheit und Ernährungssicherheit GmbH Ribo-typage PCR
US11661577B2 (en) 2007-07-26 2023-05-30 California Institute Of Technology Co-incubating confined microbial communities

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US6312903B1 (en) 1994-06-24 2001-11-06 Innogenetics N.V. Simulataneous detection, identification and differentiation of eubacterial taxa using a hybridization assay
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