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WO2002086153A1 - Determination de la resistance aux antibiotiques contenant du $g(b)-lactame par comparaison de deux genes de la $g(b)-lactamase - Google Patents

Determination de la resistance aux antibiotiques contenant du $g(b)-lactame par comparaison de deux genes de la $g(b)-lactamase Download PDF

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WO2002086153A1
WO2002086153A1 PCT/AU2002/000510 AU0200510W WO02086153A1 WO 2002086153 A1 WO2002086153 A1 WO 2002086153A1 AU 0200510 W AU0200510 W AU 0200510W WO 02086153 A1 WO02086153 A1 WO 02086153A1
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genetic sequence
esbl
lactamase
substitution
genetic
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Christopher Bruce Howard
David Scott Hammond
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Diatech Pty Ltd
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Diatech Pty Ltd
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Priority claimed from AUPR4608A external-priority patent/AUPR460801A0/en
Priority claimed from AUPR8704A external-priority patent/AUPR870401A0/en
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Publication of WO2002086153A1 publication Critical patent/WO2002086153A1/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
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer

Definitions

  • the present invention relates generally to a method for the genetic detection of microorganisms exhibiting enhanced resistance to j8-lactam-containing antibiotics.
  • the detection method further provides a risk analysis of particular biological samples or environmental locations including hospitals, or other localities in which health- compromised subjects may be situated, for a likelihood of these biological samples or environmental locations having microorganisms with enhanced resistance to /3-lactam- containing antibiotics.
  • a biological sample in this context includes a culture and more particularly a purified culture of microorganisms isolated from a biological sample.
  • the method of the present invention is based on a determination of the relative ratios of genetic material encoding an extended spectrum /3-lactamase (ESBL) compared to genetic material which either does not encode an ESBL or which does not contain a mutation conferring ESBL activity on a non-ESBL.
  • the extent of resistance is a measure of the ratio of the presence of particular nucleotide alterations in genetic sequences encoding ESBLs.
  • the method of the present invention permits high through-put screening of biological samples including microbial cultures or environmental locations or other samples for the potential for microorganisms in these samples to confer various levels of resistance to particular /3-lactam antibiotics.
  • /3-lactam drugs e.g. amoxicillin, cephalothin, clavulanate, aztreonam amongst many others
  • /3-Lactams target enzymes that are unique to bacteria and are thus highly selective. They have been widely prescribed. In the absence of resistance, /3-lactams are the first choice for treatment in 45 of 78 common bacterial infections. The evolution of resistance to these drugs has raised the cost of antibiotic therapy and reduced its effectiveness, leading to increased rates of morbidity and mortality.
  • ⁇ -lactam antibiotics inhibit bacterial cell wall biosynthesis.
  • the drugs form covalent complexes with a group of transpeptidases/carboxypeptidases called penicillin binding proteins (PBPs).
  • PBP penicillin binding proteins
  • PBP inactivation disrupts cell wall biosynthesis, leading to self-lysis and death of the bacteria.
  • Bacteria use several different mechanisms to escape from /3-lactam drugs. Probably the most widespread is the hydrolysis of /3-lactams by /3-lactamase enzymes. The persistent exposure of bacteria to antibiotic compounds has resulted in the selection of bacterial genes encoding mutant /3-lactamase enzymes that have extended substrate specificity. These genes provide bacteria with the capacity to overcome the antibiotic nature of these compounds.
  • ESBLs extended spectrum /3-lactamases
  • ESBLs are largely derived from the /3-lactamases TEM-1 or SHV-1 by one or more amino acid substitutions.
  • these genes are carried on large conjugative plasmids permitting their transfer to other bacterial hosts.
  • the method of conjugative transfer has the potential to spread these genes to a vast number of bacterial hosts and potentially create highly resistant strains ("super strains") of common bacteria.
  • DNA-based methods have been proposed to identify ESBL-encoding genetic material.
  • Previously used methods include DNA-hybridization (Cooksey et al, Antimicrob. Agents Chemother. 28: 154-156, 1985; Huovin et al, Antimicrob. Agents Chemother. 32: 175- 179, 1988; Ouellette et al, Antimicrob. Agents Chemother. 32: 397-399, 1988), oligoiyping (Mlessnesst, C. and Courvali, P., Antimicrob. Agents Chemother. 34: 2210-2216, 1990), restriction fragment length polymorphism analysis (Neusch-Inderbinen et al, Eur. J. Clin. Microbil Infect. Dis.
  • the mini-sequencing procedure or First Nucleotide Change (FNC) method (Dale et al, International Patent Application No. PCT/AU90/00058 and Australian Patent No. 656514), has previously proved effective for the diagnosis of polymorphic sites in humans (Chiang et al, J. Hypertens. 15: 607-611, 1997; Hietala et al, Clin. Chem. 42: 1398-1404, 1996; jalanko et al, Clin. Chem. 38: 39- 43, 1992; Mariotti et al, J.
  • FNC First Nucleotide Change
  • the method interrogates polymorphic sites through primer annealing immediately upstream of the polymorphic site followed by determination of the identity of a single base incorporated.
  • the instant inventors have developed a genetic assay approach for determining the likelihood of resistance of microorganisms to particular antibiotics.
  • the assay may be readily automated and permits high through-put screening of environmental and other samples including microbial cultures for potentially /3-lactam antibiotic-resistant microorganisms.
  • SEQ ID NO: Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO:).
  • the SEQ ID NOs: correspond numerically to the sequence identifiers ⁇ 400>1, ⁇ 400>2, etc.
  • a sequence listing is provided after the claims.
  • the present invention is predicated in part on the use of genetic analysis to determine the presence and, in particular, the ratio of a first genetic sequence encoding a first /3-lactamase and a second genetic sequence encoding a second /3-lactamase.
  • the second /3-lactamase may or may not have an amino acid substitution to render the second /3-lactamase having substantially the same or greater activity compared to the first /3-lactamase.
  • Reference to "greater” activity also includes a broader spectrum of activity.
  • the ratio of the first ⁇ - lactamase to the second /3-lactamase provides an indication of a microorganism exhibiting a particular resistance to a /3-lactam antibiotic or a range of a family of /3-lactam antibiotics.
  • the first and secodn genetic sequences may represent different alleles at a particular locus or may represent genetic sequences at different loci.
  • the first 3-lactamase is preferably an extended spectrum /3-lactamase (ESBL) whereas the second 3-lactamase may be an ESBL or a non-ESBL.
  • the ESBL of the second /3-lactamase may be non-ESBL carrying a mutation rendering it an ESBL.
  • the method of the present invention may also be used for identifying the level of resistance of a microorganism to a /3-lactam antibiotic by determining the ratio of first and second nucleotide sequences encoding an ESBL and non-ESBL, respectively. These first and second genetic sequences may represent different alleles at the same or different loci. In an alternative embodiment, the ratio is determined between first and second genetic sequences encoding first and second ESBLs, respectively.
  • the preferred microorganism is a species of Klebsiella such as Klebsiella pneumoniae.
  • the first nucleotide positions of codons 238 and 240 are altered to produce an ESBL with a greater resistance to a /3-lactam antibioitic when compared with a /3-lactamase where the first nucleotide positions of codons 238 and 240 are not altered.
  • the ratio of A to G at either position is indicative of an ESBL with a greater resistance to a /3-lactam antibiotic. Even more preferably the summation of the ratio of A to G at both positions is indicative of an ESBL with a greater resistance to a /3-lactam antibiotic.
  • Figure 1 is a graphical representation of Bubble charts depicting the relationships between the results of the FNC interrogations of polymorphic sites in codons 238 and 240 and the MIC values for ATM [aztreonam] (A), CAZ [ceftazidime] (B) and CTX [cefotaxime] (C).
  • the diameters of the bubbles are directly proportional to the log (100 x MIC values).
  • the values on the axes are the logs of the ratios of the means of the raw figures for incorporation of A and G at the two polymorphic sites, taken from all three replicates of the FNC assays.
  • Each bubble is labelled with the strain.
  • Figure 1 A the b/asnv genotypes as defined by sequencing and FNC are indicated.
  • Figure 2 is a bubble chart representation depicting the relationships between the results of the real-time (RT)-PCR assays and the MIC values for ATM (A); CAZ (B) and CTX (C). Each bubble represents an isolate and their diameters of the bubble are directly proportional to the log (110 x MIC values). The values on the axes are the difference between the Ct values obtained from the primers specific for WT and mutant alleles.
  • the data from the codon 238 site is plotted on the X axis and the data from codon 204 is on the Y axis.
  • Figure 3 is a graphical representation showing frequencies of G238S and E240K mutations as determined by cloning of PCR products in isolates shown in Figure 1. A summary of sequence identifiers is provided below:-
  • the present invention is predicated in part on the development of a genetic assay to determine the likelihood that a sample including a microbial culture comprises microorganisms exhibiting a certain level of resistance to a /3-lactam-containing antibiotic.
  • one aspect of the present invention contemplates a method for determining the likelihood that a sample comprises a microorganism exhibiting a particular level or spectrum of resistance to a /3-lactam-containing antibiotic, said method comprising determining the ratio of the presence of a first genetic sequence encoding a first ⁇ - lactamase relative to a second genetic sequence encoding a second /3-lactamase said second /3-lactamase has the capacity of carrying an amino acid substitution which confers substantially the same level and/or spectrum of activity as the first /3-lactamase wherein the greater the ratio of the first genetic sequence relative to the second genetic sequence is indicative of the presence of a microorganism exhibiting higher level resistance to said ⁇ - lactam-containing antibiotic than an organism with a lower ratio of a said same genetic sequences.
  • the first /3-lactamase is an extended spectrum 3-lactamase (ESBL).
  • the second /3-lactamase may be an ESBL or a non-ESBL.
  • another aspect of the present invention provides a method for identifying the level of resistance of a microorganism to a /3-lactam antibiotic, said method comprising optionally determining the presence of a first genetic sequence encoding an ESBL and then determining the ratio of the presence of said first genetic sequence and a second genetic sequence encoding a non-ESBL wherein said second genetic sequence differs from said first genetic sequence by one or more nucleotides wherein the higher the ratio between said first and second genetic sequences is indicative of an organism with a higher level of resistance to said ⁇ -lactam antibiotic.
  • the present invention contemplates a method for identifying the level of resistance of a microorganism to a /3-lactam antibiotic, said method comprising optionally determining the presence of a first genetic sequence encoding a first ESBL and then determining the ratio of the presence of said first genetic sequence and a second genetic sequence encoding a second ESBL wherein said first ESBL confers greater resistance to /3-lactam antibiotics compared to said second ESBL and wherein said second genetic sequence differs from said first genetic sequence by one or more nucleotides wherein the higher the ratio between said first and second genetic sequences is indicative of an organism with a higher level of resistance to said /3-lactam antibiotic.
  • the "presence" of a greater sequence generally means gene or plasmid copy number per cell including numbers of alleles per genetic molecule per cell.
  • /3-Lactam antibiotics contemplated by the present invention include but are not limited to a penicillin, cephalosporin or monobactams such as cefotaxime, ceftazidime, aztreonam, amoxicillin, ampicillin, azlocillin, mezlocillin, apalcillin, hetacillin, bacampicillin, carbenicillin, sulbenicillin, ticarcillin, piperacillin, mecillinam, pivmecillmam, methicillin, ciclacillin, talampicillin, aspoxicillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, nafcillin, pivampicillin, cephalothin, cephaloridine, cefaclor, cefadroxil, cefamandole, cefazolin, cephalexin, cephradine, ceftizoxime, cefoxitin, cepha
  • the present invention further provides a method for the detection of an ESBL having a particular spectrum of activity and present in a biological sample including a microorganism or culture thereof, said method comprising determining the ratio of the presence of a first genetic sequence encoding a first ESBL relative to a second genetic sequence encoding a second ESBL and wherein the activity or spectrum of the first ESBL is greater than the second ESBL wherein the greater the ratio of the first genetic sequence relative to the second genetic sequence is indicative of the presence of a first ESBL conferring a higher level of resistance to said /3-lactam-containing antibiotic compared to said second ESBL.
  • biological sample in this instance includes a culture of microorganisms isolated from another biological sample such as from a subject or environment including hospital location.
  • a particular level of resistance is relative to a non-ESBL or ESBL conferring a low level of resistance.
  • the microorganism contemplated by the present invention is generally an enterobacterium or any microorganism capable of carrying a plasmid encoding a /3-lactamase and in particular a TEM-1 or SHV-1 /3-lactamase.
  • Gram-positive and Gram- negative bacteria such as, for example, bacteria of the genus Staphylococcus (such as Staphylococcus aureus and Staphylococcus epidermis), Streptococcus (such as Streptococcus agalactine, Streptococcus penumoniae and Streptococcus faecalis), Micrococcus (such as Micrococcus luteus), Bacillus (such as Bacillus subtilis), Listerella (such as Listerella monocytogenes), Escherichia (such as Escherichia coli), Klebsiella (such as Klebsiella pneumoniae), Proteus (such as Proteus mirabilis and Proteus vulgaris), Salmonella (such as Salmonella typhosa), Shigella (such as Shigella sonnei), Enterobacter (such as Enterobacter aerogenes and Enterobacter facium), Serratia (such as Serratia marcescens), P
  • the microorganism is a Klebsiella species such as but not limited to a strain of Klebsiella pneumoniae or a related organism.
  • Another aspect of the present invention contemplates, therefore, a method for determining the likelihood that a sample comprises a bacterium of the genus Staphylococcus,
  • the present invention provides a method for identifying the level of resistance of a bacterium of the genus Staphylococcus, Streptococcus, Micrococcus, Bacillus, Listerella, Escherichia, Klebsiella, Proteus, Salmonella, Shigella, Enterobacter , Serratia, Pseudomonas, Acinetobacter, Nocardia and Mycobacterium to a 3-lactam containing antibiotic such as a penicillin, cephalosporin or monobacterium such as cefotaxime, ceftazidime, aztreonam, amoxicillin, ampicillin, azlocillin, mezlocillin, apalcillin, hetacillin, bacampicillin, carbenicillin, sulbenicillin, ticarcillin, piperacillin, mecillinam, pivmeciUinam, methicillin, ciclacillin, talampicillin, aspoxicillin,
  • Yet another embodiment of the present invention contemplates a method for identifying the level of resistance of a bacterium of the genus Staphylococcus, Streptococcus, Micrococcus, Bacillus, Listerella, Escherichia, Klebsiella, Proteus, Salmonella, Shigella, Enterobacter , Serratia, Pseudomonas, Acinetobacter, Nocardia and Mycobacterium to a /3-lactam containing antibiotic such as a penicillin, cephalosporin or monobacterium such as cefotaxime, ceftazidime, aztreonam, amoxicillin, ampicillin, azlocillin, mezlocillin, apalcillin, hetacillin, bacampicillin, carbenicillin, sulbenicillin, ticarcillin, piperacillin, mecillinam, pivmeciUinam, methicillin, ciclacillin, talampicillin, a
  • the microorganism is a Klebsiella species such as but not limited to a strain of Klebsiella pneumoniae or a related organism.
  • the present invention contemplates a method for determining the likelihood that a sample comprises a Klebsiella species such as but not limited to a strain of Klebsiella pneumoniae or a related organism such as Escherichia coli or Pseudomonas sp.
  • a Klebsiella species such as but not limited to a strain of Klebsiella pneumoniae or a related organism such as Escherichia coli or Pseudomonas sp.
  • a /3-lactam-containing antibiotic such as a penicillin, cephalosporin or monobacterium such as cefotaxime, ceftazidime, aztreonam, amoxicillin, ampicillin, azlocillin, mezlocillin, apalcillin, hetacillin, bacampicillin, carbenicillin, sulbenicillin, ticarcillin, piperacillin, mecillinam, pivmeciUinam, methicillin, ciclacillin, talampicillin, aspoxicillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, nafcillin, pivampicillin, cephalothin, cephaloridine, cefaclor, cefadroxil, cefamandole, cefazolin, cephalexin, cephradine, ceftizoxime, cefoxitin, cephacet
  • the present invention contemplates a method for identifying the level of resistance of a bacteria of the genus Klebsiella species such as but not limited to a strain of Klebsiella pneumoniae or a related organism such as Escherichia coli or Pseudomonas sp.
  • antibiotic such as a penicillin, cephalosporin or monobacterium such as cefotaxime, ceftazidime, aztreonam, amoxicillin, ampicillin, azlocillin, mezlocillin, apalcillin, hetacillin, bacampicillin, carbenicillin, sulbenicillin, ticarcillin, piperacillin, meci inam, pivmeciUinam, methicillin, ciclacillin, talampicillin, aspoxicillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, nafcillin, pivampicillin, cephalothin, cephaloridine, cefaclor, cefadroxil, cefamandole, cefazolin, cephalexin, cephradine, ceftizoxime, cefoxitin, cephacetrile, cefotiam,
  • the present invention contemplates a method for identifying the level of resistance of a bacteria of the genus Klebsiella species such as but not limited to a strain of Klebsiella pneumoniae or a related organism such as Escherichia coli or Pseudomonas sp.
  • a /3-lactam antibiotic such as a penicillin, cephalosporin or monobacteriums such as cefotaxime, ceftazidime, aztreonam, amoxicillin, ampicillin, azlocillin, mezlocillin, apalcillin, hetacillin, bacampicillin, carbenicillin, sulbenicillin, ticarcillin, piperacillin, meciUinam, pivmeciUinam, methicillin, ciclacillin, talampicillin, aspoxicillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin, nafcillin, pivampicillin, cephalothin, cephaloridine, cefaclor, cefadroxil, cefamandole, cefazolin, cephalexin, cephradine, ceftizoxime, cefoxitin, cephacetrile, cefotiam,
  • the first and second genetic sequences may be both located on a plasmid or one may be plasmid borne and the other located on the chromosome. Generally, the first genetic sequence encoding an ESBL is plasmid borne. The first and second genetic sequences may also be different alleles at the same loci.
  • the present invention is predicated in part on the use of genetic analysis to determine the presence and in particular the ratio of a first genetic sequence encoding an ESBL and a second genetic sequence encoding either an ESBL or non-ESBL in a microorganism.
  • the level of risk that a microorganism is highly resistant to /3-lactam antibiotics is considered high when the ratio of first to second genetic sequences is high such as greater than 0.5 and more preferably greater than 1.0 including greater than 5.
  • a microorganism comprising two or more genetic sequences each encoding an ESBL may also be assessed for a level of resistance based on a ratio of mutations wherein each mutation confers ESBL status to a non-ESBL.
  • the copy number per microorganism of the first genetic sequence encoding an ESBL may be considered as M.
  • the copy number of the second genetic sequence may be representated as N. Consequently, the present invention preferably provides a high level of risk that a microorganism is resistant to a wide range of 3-lactam antibiotics when:
  • the present invention contemplates, therefore, a copy number dependence on the level of resistance conferred by an ESBL.
  • the first allele corresponding to a first genetic sequence may be represented as X having copy number M.
  • a second allele corresponding to a second genetic sequence may be represented as Y having copy number N.
  • Each allele X, Y, etc. may comprise one of four different nucleotides, i.e. A, T, C or G. At least one nucleotide in each allele confers ESBL activity. Consequently, there may be two enquiries which can be undertaken.
  • the ratio — can be determined.
  • each allele may be defined by the general formula: -
  • A is a particular allele such as X or Y;
  • p represents the nucleotide sequence defining the alle conferring ESBL activity
  • p 1 represents the nucleotide sequence defining the allele having non-ESBL activity or less than conferred by p.
  • the extent of ESBL activity can be defined by Ap > Ap 1 .
  • the ratio of each allele can then be determined such as — - and
  • a given allele will confer ESBL activity when — ⁇ and/or — - are greater
  • the second enquiry may be made independently of the first enquiry or in conjunction with it.
  • the ESBL carries a nucleotide substitution at codon number 238 and/or 240 resulting in an amino acid substitution.
  • G ⁇ A substitution at codon 238 results in a glycine to serine substitution, and is indicative of the presence of the bla ⁇ sv-2a ESBL allele, the presence of which provides a /3-lactamase allele with extended substrate specificity.
  • an ESBL allele may contain a second substitution of G ⁇ A at codon 240 resulting in a glutamate ⁇ lysine substitution, such an allele is represented by the bla SHV - ⁇ allele.
  • an ESBL may contain a substitution at position 238 and/or 240 and it is known that an ESBL containing both substitutions exhibit a greater resistance to /3-lactam antibiotics.
  • Amino acid or codon positions are as determined by Ambler et al, Biochem. J. 256: 269-270, 1991.
  • a substitution to glycine at position 238 may undergo a further substitution to alanine. Some substitutions at other locations may also cause resistance to inhibitors of ⁇ - lactam antibiotics, therefore, affecting the level of resistance or sensitivity to /3-lactam antibiotics.
  • One example contemplated by the present invention is a serine ⁇ glycine change at residue 130.
  • Yet another mutation contemplated by the present invention is an asparagine ⁇ serine substiutution at codon 71 and or a glycine ⁇ aspartate at codon 176 and/or a tyrosine ⁇ lysine or serine at codon 144.
  • the present invention is directed to all members of the /3-lactamase gene family including members of the SHV gene family.
  • Other /3-lactamase gene families include the TEM and OXA gene families. While not intending to limit the scope of the present invention, it is known that in the TEM family of /3-lactamases, a glutamate ⁇ lysine alteration at residue 104, a methionine to threonine substitution at residue 182 and a glycine ⁇ serine substitution at residue 238 individually or collectively result in a /3-lactamase enzyme which has an extended substrate specificity.
  • an allele refers to a derivative, variant or isoform of a genetic sequence comprising a nucleotide substitution at a given position within the genetic sequence.
  • an ESBL may contain a number of substitutions which each confer a different substrate specificity. As such, each substitution when considered separately or together may be considered an individual allele of an ESBL.
  • Preferred alleles of ESBL include but are not limited to bla SHV -2E and bla SHV- ⁇ -
  • one aspect of the present invention contemplates a method for determining the likelihood that a sample comprises a Klebsiella species such as but not limited to a strain of Klebsiella pneumoniae or a related organism such as Escherichia coli or Pseudomonas sp.
  • a Klebsiella species such as but not limited to a strain of Klebsiella pneumoniae or a related organism such as Escherichia coli or Pseudomonas sp.
  • said method comprising determining the ratio of the presence of a first genetic sequence encoding an first /3-lactamase having a serine and/or a lysine at amino acid positions 238 and 240, respectively relative to a second genetic sequence encoding a second /3-lactamase having a glycine and/or glutamate at amino acid positions 238 and 240, respectively wherein an amino acid substitution in the second /3-lactamase confers substantially the same spectrum and or level of the activity as the first /3-lactamase wherein the greater the ratio of the first genetic sequence relative to the second genetic sequence is indicative of the presence of a Klebsiella species or a related organism exhibiting higher level resistance to said /3-lactam-containing antibiotic than an organism with a lower ratio of a said same genetic sequences.
  • the present invention provides a method for identifying the level of resistance of a bacteria of the genus Klebsiella species such as but not limited to a strain of Klebsiella pneumoniae or a related organism such as Escherichia coli or Pseudomonas sp.
  • said method comprising determining the presence of a first genetic sequence encoding an ESBL having a serine and/or a lysine at amino acid positions 238 and 240, respectively relative to a second genetic sequence encoding a non-ESBL having a glycine and/or glutamate at amino acid positions 238 and 240, respectively wherein the higher the ratio between said first and second genetic sequences is indicative of an bacteria of the genus Klebsiella species or a related organism with a higher level of resistance to said /3-lactam antibiotic compared to an organism with a lower ratio of first to second genetic sequences.
  • the present invention contemplates a method for identifying the level of resistance of a bacteria of the genus Klebsiella species such as but not limited to a strain of Klebsiella pneumoniae or a related organism such as Escherichia coli or Pseudomonas sp.
  • said method comprising dete ⁇ nining the presence of a first genetic sequence encoding a first ESBL having a serine and/or a lysine at amino acid positions 238 and 240, respectively relative to a second genetic sequence encoding a second ESBL having a glycine and/or glutamate at amino acid positions 238 and 240, respectively wherein the higher the ratio between said first and second genetic sequences is indicative of an organism with a higher level of resistance to said /3-lactam antibiotic.
  • Nucleotide substitutions in gene sequences may result in an altered activity of an enzyme through increased substrate binding, substrate product release, or enhanced catalytic activity of the substituted enzyme.
  • the substituted /3-lactamase of the present invention is a /3-lactamase with an extended substrate range (i.e. ESBL).
  • another aspect of the present invention contemplates a method for determining the likelihood that a sample comprises a Klebsiella species such as but not limited to a strain of Klebsiella pneumoniae or a related organism such as Escherichia coli or Pseudomonas sp.
  • a Klebsiella species such as but not limited to a strain of Klebsiella pneumoniae or a related organism such as Escherichia coli or Pseudomonas sp.
  • said method comprising determining the ratio of nucleotides A, C, G or T at the first position or second position of a codon in a first /3-lactamase present in said Klebsiella species which exhibits a level of resistance to a /3-lactam antibiotic and determining the ratio of nucleotides A, C, G or T at a first or second position of another codon in a second /3-lactamase present in said Klebsiella species or a related organism which also exhibits a level of resistance to a /3-lactam antibiotic wherein an amino acid substitution in the second /3-lactamase confers substantially the same spectrum and or level of the activity as said first /3-lactamase wherein the greater the ratio of the first genetic sequence relative to the second genetic sequence is indicative of the presence of a Klebsiella species or related organism having a higher level of resistance to said /3-lactam- containing antibiotic, said method comprising determining the ratio of nucleotides A, C,
  • the first nucleotide positions of codons 238 and 240 are altered to produce an ESBL with a greater resistance to a /3-lactam antibioitic when compared with a /3-lactamase where the first nucleotide positions of codons 238 and 240 are not altered.
  • the ratio of A to G at either position is indicative of an ESBL with a greater resistance to a /3-lactam antibiotic. Even more preferably the summation of the ratio of A to G at both positions is indicative of an ESBL with a greater resistance to a /3-lactam antibiotic.
  • the present invention provides a method for detecting the presence of a ESBL in a Klebsiella species or a related organism which exhibits a level of resistance to a ⁇ - lactam antibiotic, said method comprising determining the ratio of A to G at the first position of codon 238 in a /3-lactamase present in a Klebsiella species or a related organism such as Escherichia coli or Pseudomonas sp.
  • the present invention provides a method identifying a Klebsiella species or a related organism such as Escherichia coli or Pseudomonas sp. comprising an ESBL which exhibits a level of resistance to a 3-lactam antibiotic said method comprising determining the ratio of A to G at the first position of codon 238 in a ⁇ - lactamase present in a Klebsiella species or a related organism which exhibits a level of resistance to a /3-lactam antibiotic and determining the ratio of A to G at the first position of codon 240 Klebsiella species or a related organism which exhibits a level of resistance to a /3-lactam antibiotic wherein the summation of the ratio's of A to G at the first nucleotide positions of 238 and 240 is indicative of a ESBL with either a single A substitution at either position 238 or 240, or an A substitution at both 238 and 240 positions, wherein a ratio of
  • a glycine at position 238 may be further modified to an alanine. Such a further substitution is encompassed by the present invention.
  • this method can also be used to determine the degree of /3-lactamase inhibitor exhibited by an organism.
  • mutations causing inhibitor resistance are the target.
  • Such mutations are typified but limited to those that cause a serine ⁇ glycine change at position 130 in SHV /3-lactamases.
  • One such inhibitor is clavulanic acid. This substitution may also form part of a genetic analysis.
  • the sample may be an environmental sample such as a hospital or particular location within a hospital or other locality in which health-compromised subjects reside. Examples of particular locations include operating theatres, intensive care wards, general wards, casualty areas, laundries, trease areas, ambulances and laboratories.
  • the sample may also be a biological sample such as a fecal sample, fecal-containing sample, specimen, biopsy and body fluid sample (e.g. blood, sputam, lymph, bile, respiratory fluid).
  • a biological sample may also be a culture of a microorganism and generally is a purified culture of a microorganism isolated from another biological source or sample.
  • the presence of the first and second genetic sequences may be detected by any convenient means including FNC, DNA hybridization, digotyping, restriction fragment length polymorphism analysis, PCR, PCR-single stranded conformation polymorphism, ligase chain reaction and real-time PCR (RT-PCR).
  • FNC and RT-PCR are particularly preferred for the practice of the present invention. These processes can be used to determine plasmid or gene copy number or alleles per genetic sequence or per cell.
  • the present invention also extends to non-genetic means such as differential immunological interaction.
  • the present invention provides an array of oligonucleotides specific to a first genetic sequence and an array of oligonucleotides specific to a second genetic sequence wherein the arrays may be located on the same or different solid supports such as biochips.
  • a biocbip employs microchip technology to permit the generation of antibody patterns to detect a particular microorganisms or oligonucleotide patterns for conducting a genetic analysis. This permits automation and/or computer analysis.
  • a "microchip” includes a matrix support comprising an array of adapter molecules, ligands or potential binding partners. The arrays are arranged such that one sample is capable of being tested using oligonucleotides specific for both first and second genetic sequences. The data obtained enables the calculation of a ratio of first to second genetic sequences.
  • the method for conducting the subject identification of the present invention is readily adapted for automation.
  • robotic systems may be used to deliver appropriate amounts of microorganisms or other samples or reagents to a solid support such as a nitrocellulose film or microtitre plate.
  • the present invention further contemplates the use of an antibody array to capture particular microorganisms from a sample which captured microrganisms are then subjected to detection means for the presence of ESBL-encoding material.
  • the present invention further comprises a kit useful for detecting or identifying particular ESBLs in a sample including a microorganism, said kit comprising an assay device comprising an array of immobilized binding partners such as antibodies or oligonucleotides useful to capture target microorganisms or to undertake genetic analysis.
  • the kit is conveniently in compartmental form and may further comprise compartments adapted to confer reagents.
  • the kit or assay device therein is also conveniently adapted for automation or for computer-assisted reading.
  • the method of this aspect of the present invention can at least be partly implemented using a suitably programmed computer.
  • the preferred data processing means comprises a suitably programmed computer and the steps of the method are preferably performed using the suitably programmed computer.
  • the input information may take the form of values, identifiers or other data in respect of the identity of interaction with the immobilized molecules.
  • the input data may be digitized.
  • a dedicated Fat Fourier transform chip can be employed as at least part of the processing means.
  • the input program and general methodology of this aspect of the present invention are useful in a range of applications including the rapid diagnosis of disease conditions, testing the efficacy of a therapeutic regimen and/or assessing the overall health of a subject as well as risk analysis of a subject or environmental location or of a biological sample.
  • the present invention extends to a method of treatment based on determining the likelihood of the presence of an antibiotic resistant Klebsiella species or related organism. Depending on the results obtained, an antibiotic or selection of antibiotics or one or more non-antibiotic chemicals may be chosen for use. This approach helps reduce the non- judicial use of antibiotics or other chemicals which can result in development of resistant strains.
  • Klebsiella pneumoniae isolates used in this study have been previously described by Schooneveldt and colleagues (Schooneveldt et al, Pathology 30: 164-168, 1998). Sixteen of the 21 strains were found to express ESBLs as defined by the double-disk diffusion test (Jarlier et al, Rev. Infect. Dis. 10: 867-878, 1988). This was supported by the MICs for cefotaxime, ceftazidime and aztreonam.
  • Escherichia coli JC411 which carries the blasuv that encodes an ESBL (Mulgrave et al, Pathology 25: 71-75, 1993) was also used.
  • AU strains were cultured in Luria-Bertani Broth and stored in cryo vials with 12% w/v glycerol at -80°C.
  • Plasmid DNA was extracted from 1.5 ml or 2.5 ml cultures grown overnight in L-broth using alkaline lysis minipreps (Maniatis et al, Molecular Cloning - A Laboratory Manual, 2 nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbour, NY, USA).
  • Overlapping regions of the genes were PCR amplified using primers which target either coding sequence (SHV-F: nucleotide position 311 to 328 TCAGCGAAA AACACCTT G [SEQ ID NO:l] and SHV-R: nucleotide position 782 to 764, TCCCGCAGATAAATCACCA [SEQ ID NO:2]) (M'Zali et al, 1996, supra) or, sites adjacent to the coding sequence (BIG-F: nucleotide position -72 to -52 CGCCGGGTTATTCTT ATTTG [SEQ ID NO:3] and BIG-R: nucleotide position +78 to +54 TCTTTCCGATGCCGCCGC CAGTCA [SEQ ID NO:4].
  • PCR reactions were carried out in 1.5 mM MgCl , 50 mM KC1, and 10 mM Tris-HCl pH 8.3 (Boehringer Mannheim, Germany), 400 ⁇ M dNTPs (Boehringer Mannheim, Germany), 20 pmoles of each primer (Gibco-Life Technologies, USA), 100 ng of plasmid DNA template and limit of Taq Polymerase (Boehringer Mannheim, Germany). After a 5 min denaturation at 96°C, 35 cycles of temperatures 96°C, 55°C and 72°C and a final extension step of 72°C for 3 mins were performed.
  • the length of the temperature steps for the 35 cycles were 30 sec each for the coding sequence primers and 1 min each for the primers adjacent to the coding sequence.
  • dimethoxyl sulphoxide final concentration 5% was added when the BIG-F primer was used.
  • Amplicons generated with the coding primers from the clinical isolates D, G, R and S and U were cloned using the pGEMt plasmid kit (Promega, USA) and JM109 High Efficiency competent Escherichia coli cells (Promega, USA). 10 to 20 clones were selected for each isolate and secondary PCR products were generated from these clones for sequencing and FNC analysis.
  • PCR products were purified using Wizard mini-columns (Promega, USA). 50-100 ng (10 ng/100 bases) of amplicon was sequenced using 3.2 pmoles of the amplification primer. The products were analyzed on an ABI 373A DNA automated Sequencer. Sequence results were aligned using the Clustal W program (Higgins et al, Nucliec Acids Res. 22: 4673- 4680, 1994.) which was accessed through the Australian National Genome Information Service (ANGIS).
  • Clustal W program Higgins et al, Nucliec Acids Res. 22: 4673- 4680, 1994.
  • ANGIS Australian National Genome Information Service
  • FNC (Dale et al, supra) is- a microtitre plate-based mini-sequencing (single nucleotide extension) assay.
  • a biotinylated primer is hybridized with a PCR generated target at a sequence one base upstream from a polymorphic site. This complex is captured by streptavidin-coated microtitre wells.
  • the primer is extended by DNA polymerase in the presence of a single fluoresceinated dideoxynucleotide (ddNTP) and unlabelled ddNTPs.
  • Bound fluoroscein is detected by enzyme-linked immuno-absorbant assay (ELISA). The assay is carried out in four wells, each with a different fiuorosceinated ddNTP.
  • the extension reactions were carried out in Sequenase buffer, 0.4 mM dithiothrietol (DTT), 0.4 mM MnCl 2 , 0.7 mM Na isocitrate, 0.015% v/v Tween 20, 0.015% v/v Nonidet.
  • MSP-238 5' Biotin-GTTTATCGCCGATAAGACC GGAGCT [SEQ LD NO: 5] was designed to detect the G to A change at position 1 of codon 238 indicative of ESBLs.
  • a procedure for interrogating the polymorphic nucleotides at the first positions of codons 238 and 240 was developed. At both positions, the presence of a G is indicative of the ancestral form while A is indicative of the extended spectrum of activity.
  • the mutation in codon 238 converts a non-ESBL to an ESBL while a second mutation in codon 240 confers very high activity against the extended spectrum substrates. Subsequent to optimisation of the procedure, the reliability of the method was assessed by generating and assaying three times the primary amplicons from all strains and retaining all the results.
  • the primers of the PCR were designed to interrogate the first positions of codons 238 and 240 and to provide PCR products of 134 bp (codon 238 interrogation) and 132 bp (codon 240 interrogation).
  • the PCRs were performed in 0.2 mL microtubes with a total reaction volume of 25 ⁇ l.
  • Each PCR reaction contained 200 ⁇ M of each dNTP (0.5 ⁇ l of 10 mM stock), 2.5 ⁇ M MgCl 2 (2.5 ⁇ l of 50 mM stock), 2.5 ⁇ l of 10X PCR buffer, 0.5 U of Platinum Taq DNA polymerase (Life Technologies), 0.5X Sybr-Green Dye (0.5 ⁇ l 25X stock), 5 pmol of the common primer and either 2.5 pmol of one of the WT specific primers or 5 pmol of one of the WT specific primers and 1 ⁇ l of 10-fold diluted bacterial cell lysate.
  • thermocycling conditions were as follows :-
  • Steps 2-4 were repeated 45 times with fluorescence measured at an excitation wavelength of 497 nm and fluorescence detection at 510 nm (Gain level 6) at the end of step 4.
  • the Ct values obtained for each isolate are shown in Table 3.
  • the Ct values are the amplification cycle at which product accumulation as measured by Sybr green fluorescence reaches a threshold value.
  • the Ct is a function of the amount of target present at the beginning of the reaction.
  • the threshold values were the default as calculated by the software provided with the Rotor-gene 2000 by the manufacturer.
  • the data show that the inventors have developed a s single-step real-time AS-PCR based method for measuring the relative copy numbers of gene encoding SHV /3-lactamase variants.

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Abstract

L'invention porte sur un procédé génétique de détermination de la résistance aux antibiotiques contenant du β-lactame présents dans des micro-organismes. Ledit procédé consiste à déterminer le rapport entre d'une part le matériau génétique codant pour une β- lactamase à spectre large (ESBL), et d'autre part le matériau génétique ne codant pas pour une ESBL, ou ne contenant pas de mutation ne conférant pas à une non ESBL l'activité d'une ESBL, ou présentant des altérations de nucléotides particuliers des séquences génétiques codant pour les ESBL.
PCT/AU2002/000510 2001-04-24 2002-04-23 Determination de la resistance aux antibiotiques contenant du $g(b)-lactame par comparaison de deux genes de la $g(b)-lactamase Ceased WO2002086153A1 (fr)

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AUPR4608A AUPR460801A0 (en) 2001-04-24 2001-04-24 A method of detection
AUPR4608 2001-04-24
AUPR8704 2001-11-07
AUPR8704A AUPR870401A0 (en) 2001-11-07 2001-11-07 A method of detection - II

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7045291B2 (en) 2002-05-17 2006-05-16 Creighton University Multiplex PCR for the detection of AmpC beta-lactamase genes
CN100413962C (zh) * 2005-06-16 2008-08-27 华中科技大学同济医学院附属同济医院 一种作用于产超广谱β-内酰胺酶大肠杆菌噬菌体
US7476520B2 (en) 1998-09-28 2009-01-13 Creighton University Primers for use in detecting beta-lactamases
CN1644710B (zh) * 2003-09-29 2010-09-15 艾本德股份有限公司 微生物抗生素抗性检测方法
WO2011138402A1 (fr) * 2010-05-05 2011-11-10 Check-Points Holding B.V. Dosages, compositions et méthodes de détection de micro-organismes pharmacorésistants
WO2017012660A1 (fr) * 2015-07-22 2017-01-26 Curetis Gmbh Test génétique pour prédire la résistance de l'espèce serratia à des agents antimicrobiens
CN108531629A (zh) * 2018-05-31 2018-09-14 广西壮族自治区兽医研究所 一种快速检测肺炎克雷伯氏菌的pcr扩增引物及其应用

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Title
BOU G. ET AL.: "Molecular characterization of FOX-4, a new AmpC-type plasmid-mediated beta-lactamase from an escherichia coli strain isolated in Spain", ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, vol. 44, no. 9, 2000, pages 2549 - 2553 *
ESSACK S.Y. ET AL.: "Complexity and diversity of Klebsiella pneumoniae strains with extended-spectrum beta-lactamases isolated in 1994 and 1996 at a teaching hospital in Durban, South Africa", ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, vol. 45, no. 1, 2001, pages 88 - 95 *
TIMM J. ET AL.: "Transcription and expression analysis, using lacZ and phoA gene fusions, of mycobacterium fortuitum beta-lactamase genes cloned from a natural isolate and a high-level beta-lactamase producer", MOLECULAR MICROBIOLOGY, vol. 12, no. 3, 1994, pages 491 - 504 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7476520B2 (en) 1998-09-28 2009-01-13 Creighton University Primers for use in detecting beta-lactamases
US7045291B2 (en) 2002-05-17 2006-05-16 Creighton University Multiplex PCR for the detection of AmpC beta-lactamase genes
US7521547B2 (en) 2002-05-17 2009-04-21 Creighton University Multiplex PCR for the detection of AmpC beta-lactamase genes
CN1644710B (zh) * 2003-09-29 2010-09-15 艾本德股份有限公司 微生物抗生素抗性检测方法
CN100413962C (zh) * 2005-06-16 2008-08-27 华中科技大学同济医学院附属同济医院 一种作用于产超广谱β-内酰胺酶大肠杆菌噬菌体
WO2011138402A1 (fr) * 2010-05-05 2011-11-10 Check-Points Holding B.V. Dosages, compositions et méthodes de détection de micro-organismes pharmacorésistants
WO2017012660A1 (fr) * 2015-07-22 2017-01-26 Curetis Gmbh Test génétique pour prédire la résistance de l'espèce serratia à des agents antimicrobiens
WO2017013220A3 (fr) * 2015-07-22 2017-03-02 Curetis Gmbh Test génétique permettant de prédire la résistance d'espèces de serratia à des agents antimicrobiens
CN108271399A (zh) * 2015-07-22 2018-07-10 阿瑞斯遗传股份有限公司 用于预测沙雷氏菌属物种对抗微生物剂的抗性的基因测试
CN108531629A (zh) * 2018-05-31 2018-09-14 广西壮族自治区兽医研究所 一种快速检测肺炎克雷伯氏菌的pcr扩增引物及其应用

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