JP6378153B2 - Methods for characterizing antibiotic resistance in microorganisms - Google Patents

Description

  The present invention relates to the field of microbial diagnosis, in particular to methods for characterizing the antibiotic resistance of microorganisms.

  Antibiotic resistance is the ability of microorganisms to withstand the effects of antibiotics. This resistance is formed by genetic mutation and plasmid exchange between microorganisms. Antibiotic resistance has already had a major impact on medicine and will continue to increase.

  A group of opportunistic microorganisms that has recently attracted new interest as exhibiting antibiotic resistance is the Enterobacteriaceae family. This bacterial species (including, for example, Klebsiella spp and Escherichia coli) includes, for example, opportunistic pathogens associated with urinary tract infections, sepsis, respiratory tract infections and diarrhea. It has been known for 30 years that these species are resistant to third-generation cephalosporins, such as oxyimino beta-lactams, but since then, examples of resistance have increased exponentially. . The strain acquires resistance by producing so-called substrate-specific extended beta-lactamase (ESBL). This ESBL is molecular class A and can inactivate third generation cephalosporins (ceftazidime, cefotaxime, cefpodoxime) and monobactam (azutreonam). ESBL is a derivative of common beta-lactamases (eg TEM and SHV beta-lactamases) in which one or more amino acids near the active site of the enzyme are substituted, and this substitution has affinity for third generation cephalosporins and monobactams And hydrolytic activity increases. Overuse of a new generation of cephalosporins will facilitate the evolution of new ESBL coverage increases. ESBL is encoded by a transmissible conjugative plasmid that is responsible for transmitting resistance to other types of Gram-negative bacteria.

  ESBL is classified into over 450 species based on physical properties and is inhibited to varying degrees by clavulanic acid, sulbactam, tazobactam, and this property is used to detect them in the laboratory. Currently, only phenotypic ESBL detection tests are used in clinical microbial tests. Molecular (genotype) testing is under development. The problem with molecular testing here is that there is no 100% correlation between genotype and phenotype. Thus, there is a limit to predicting molecular test values for any bacterial phenotype, including ESBL-producing bacteria.

  In general, current phenotyping tests are more sensitive and specific than ESBL genotyping tests. All phenotypic ESBL detection tests are based on the same principle. That is, changes in inhibition of bacterial growth in the presence of beta-lactam antibiotics or in the presence of a combination of beta-lactam antibiotics and beta-lactamase inhibitors are evaluated. Various manual inspection and automation platforms are commercially available for performing such phenotypic tests. Manual testing uses discs or strips impregnated with beta-lactam antibiotics or a combination of beta-lactam antibiotics and beta-lactamase inhibitors. This impregnated material is placed on a solid medium previously inoculated with a bacterial suspension at a known concentration. After overnight incubation, growth inhibition can be determined visually and quantified based on the diameter of the inhibition zone. Automated systems are also based on measuring bacterial growth at various concentrations in the presence of a panel of beta-lactam antibiotics or a combination of beta-lactam antibiotics and beta-lactamase inhibitors. Such system results are obtained after 4-18 hours.

  Currently, there is a need for means and methods that can more quickly diagnose ESBL-producing bacteria. There is also a need for ESBL detection tests that can be used to enzymatically characterize ESBL enzymes in bacterial clinical tests to track evolutionary trends and to assess and predict effective doses of antibiotic treatment. To do. Preferably, such means will generally be widely applicable to the characterization of microbial antibiotic resistance.

  The present invention now provides a method for the rapid diagnosis of antibiotic-modifying enzyme-producing microorganisms, in particular microorganisms producing ESBL (in a preferred embodiment). The present invention further provides a method for characterizing the antibiotic modifying enzyme itself. Such characterization can be useful for early detection of new types of resistance.

In a first aspect, the present invention provides a method for characterizing antibiotic resistance of a microorganism, the method comprising the following steps:
(A) providing a reference mass spectrum of the molecular target of the antimicrobial compound compound;
(B) providing an exposed sample by exposing the microorganism to the antimicrobial compound in an aqueous liquid;
(C) obtaining a mass spectrum of the exposed sample;
(D) comparing the mass spectrum acquired in step (c) with the reference mass spectrum of step (a);
(E) after the exposure, it is determined from the comparison whether a modification of the chemical composition of the molecular target or an overproduction of the molecular target has occurred, and the modification or the overproduction is observed Confirming that the microorganism is potentially resistant to the antimicrobial compound.

  In a preferred embodiment of the method, the modification includes enzymatic inactivation or enzymatic degradation of the antimicrobial compound and / or methylation or overproduction of its molecular target. More preferably, this enzymatic degradation is by degradation with beta-lactamase. In such cases, the antimicrobial compound can be a beta-lactam antibiotic, or any other beta-lactamase substrate. Thus, in another preferred embodiment of the method, the antimicrobial compound can be replaced by a substrate compound of an enzyme that modifies the antimicrobial compound. In another preferred embodiment, the antimicrobial compound is a beta-lactam antibiotic, preferably selected from the group consisting of penicillin, cephalosporin, cephamycin, and carbapenem, more preferably ceftazidime, cefotaxime, ceftriaxone. , Cefpodoxime, and aztreonam.

  It is clear that depending on the mechanism of antibiotic resistance, the molecular target (eg folic acid) of the antibacterial compound may also be over-produced, resulting in resistance to folic acid antagonists. Target overproduction can be detected by using an internal standard and observing an increase in the target / internal standard ratio. A suitable internal standard may be a nucleic acid such as DNA.

  Furthermore, depending on the mechanism of antibiotic resistance, methylation of the molecular target (eg nucleic acid) of the antibacterial compound may be detectable.

  In yet another preferred embodiment of the method of the invention, the method is performed by exposing the microorganism to a plurality of antimicrobial compounds simultaneously, thereby characterizing the antibiotic resistance of the microorganism for a plurality of antibiotic compounds. Is done.

  In yet another preferred embodiment of the method of the present invention, the enzymatic inactivation or enzymatic degradation of the antimicrobial compound is caused by beta-lactamase. In particularly preferred embodiments, the beta-lactamase enzyme may be selected from the group consisting of cephalosporinase (including substrate-specific extended cephalosporinase), penicillinase, carbenicillinase, cloxacillinase, and carbapenemase.

  In an even more preferred embodiment of the method of the present invention, the beta-lactamase enzyme is a substrate-specific extended beta-lactamase (ESBL).

  In a still further preferred embodiment of said method, the microorganism is a microorganism suspected of producing ESBL, preferably a gram negative bacterium, more preferably Klebsiella pneumoniae, Escherichia coli, Klebsiella It is a gram-negative bacterium selected from the group consisting of Klebsiella oxytoca and Proteus mirabilis.

  Samples used in embodiments of the present invention include microorganisms or their lytic organisms. The microbial sample can be a sample of a microbial culture. Such a culture may not be a pure culture. Alternatively, media fractions or direct clinical material can also be sample sources.

  In yet another preferred embodiment of the method, the method is part of a method for the characterization of a microbial antibiotic modifying enzyme, preferably the antibiotic modifying enzyme is a substrate specificity extended form. It is a beta-lactamase (ESBL) enzyme. In one particularly preferred embodiment, the method of the invention is part of a method for characterization of a substrate-specific extended beta-lactamase (ESBL) enzyme.

  Preferably, the method for characterization of the enzyme according to the present invention includes a rate of modification (preferably degradation) of the antimicrobial compound or the substrate compound in the presence or absence of a specific enzyme inhibitor, and Or determining the rate of production of enzymatic modification products of the compound or substrate, or the rate of overproduction of the molecular target of the compound, whereby the Michaelis-Menten (Km) constant of the enzyme and Determine the maximum reaction rate (Vmax).

  In yet another preferred embodiment of the method, the mass spectrum is acquired using MALDI triple quadrupole mass spectrometry.

  In yet another preferred embodiment of the method, the exposed sample is an exposed unpurified cell lysate of the microorganism.

  In yet another preferred embodiment of the method, the method further comprises the step of quantifying the microorganism. Preferably, the microorganism is quantified in the sample by quantifying one or more structural biomolecules or metabolites derived from the microorganism. In a preferred embodiment, the structural biomolecule or metabolite is selected from the group consisting of nucleic acids, preferably (genetic) DNA. DNA exists as a single molecule in the cell and can be quantified using, for example, PCR or DNA probe mediated technology.

In yet another aspect, the present invention provides a kit of parts for characterizing microbial beta-lactam antibiotic resistance comprising:
a) a lysis buffer for lysing microorganisms;
b) at least one antibacterial compound, or a substrate for an antibacterial compound modifying enzyme, and c) a MALDI matrix material,
The parts kit further includes:
d) A carrier carrying said at least one antimicrobial compound or substrate, said carrier optionally in the form of a disposable mass spectral sample support.

In yet another aspect, the present invention provides a system adapted to characterize the beta-lactam antibiotic resistance of a microorganism by the method of the present invention as described above, said system comprising one or more of the following: :
At least one antibacterial compound, or a substrate for an antibacterial compound modifying enzyme,
A container for exposing a microorganism, its cell lysate, or its culture supernatant to at least one antimicrobial compound in an aqueous liquid, preferably said at least one substrate compound is provided in said container To be
-A lysis buffer for lysing said microorganisms;
-A MALDI matrix material,
-Mass spectrometer,
A reference mass spectrum of the antibacterial compound, its enzymatic modification product, its molecular target, or a substrate compound of the modified enzyme,
-Sample support for mass spectra,
Optionally further includes:
-An automated pipettor for liquid handling,
A computer program comprising computer program code means for carrying out all the steps of the method of the invention described above when the program runs on a computer, for example algorithms for interpreting results, interface software, expert systems Includes software.

  In another aspect, the present invention provides a computer program comprising computer program code means for performing all the steps of the method of the present invention described above when the program runs on a computer.

  The invention, in another aspect, includes a computer program code means stored on a computer readable medium for performing the above-described method of the invention when the program product runs on a computer. Providing products.

DEFINITIONS The terms “antibiotic” and “antibiotic compound” are used interchangeably herein and herein reduce the viability of bacteria or inhibit the growth or reproduction of bacteria, Refers to a compound or composition. “Inhibiting growth or reproduction” means that the generation cycle increases at least 2-fold, preferably at least 10-fold, preferably at least 100-fold, most preferably indefinitely whole cell death Means that. As used in this disclosure, antibiotics are further intended to include antibacterial agents, bacterial growth inhibitors, or bactericides. Non-limiting examples of antibiotics useful in embodiments of the invention include penicillin, cephalosporin, aminoglycosides, sulfonamides, macrolides, tetracyclines, lincosamides, quinolones, chloramphenicol, glycopeptides, metronidazole, rifamupine, Isoniazid, spectinomycin, folic acid inhibitor, sulfamethoxazole and the like.

  The term “beta-lactam antibiotic” is used to indicate a compound with antibiotic properties that contains a beta-lactam functional group. A beta-lactam ring (β-lactam) is a cyclic amide containing a heteroatom ring structure and consists of three carbon atoms and one nitrogen atom. Non-limiting examples of beta-lactam antibiotics useful in embodiments of the invention include penicillin, cephalosporin, cephamycin, penem, carbapenem, monobactam. Beta-lactam antibiotics are effective against a wide range of bacterial infections (in the absence of resistance). The term “beta-lactam antibiotic” as used herein is considered to include those that have undergone some mass or structural change due to inactivation by antibiotic-resistant microorganisms. Here, such a mass change or structural change can be detected by a mass spectrum.

  The term `` third generation cephalosporin '' includes cefixime, ceftazidime, cefotaxime, ceftriaxone, cefcapene, cefdaroxime, cefdinir, cefditorene, cefetameth, cefmenoxime, cefodizime, cefoperazone, cefotaxime, cefpimazole, cefopidem , Ceftiolen, ceftizoxime, oxacephem and the like, but are not limited thereto.

  The term “beta-lactamase” means an enzyme (EC 3.5.2.6) produced by microorganisms, preferably bacteria, that has the ability to hydrolyze the beta-lactam ring of a beta-lactam antibiotic. Such enzymes are often classified into four major classes (Class A, B, C, and D) according to the so-called Ambler classification scheme, which is mainly based on protein homology. Examples of beta-lactamases include cephalosporinase, penicillinase, carbenicillinase, cloxacillinase, carbapenemase, and ceftazidimase. The meaning of this term includes “normal” beta-lactamases, substrate-specific extended beta-lactamases (ESBL), and AmpC beta-lactamases. Preferred beta-lactamases in embodiments of the present invention are group A and D beta-lactamase enzymes according to the Ambler classification, and also beta-lactam enzymes belonging to group 2 according to the Bush classification (Bush et al. 1995. Antimicrob Agents Chemother. 39: 1211-33). Ambler class A antibiotics are classical active site serine beta-lactamases, class D is a specific group of serine beta-lactamases, and has little sequence similarity to class A beta-lactamases and OXA (oxacillinase) Well known as a group. Metallocarbapenemase is also preferable.

  The term “substrate-specific extended beta-lactamase” (abbreviated as ESBL), as used herein, was originally called “extended broad-spectrum beta-lactamase” and is capable of hydrolyzing oxyiminocephalosporin. Formed as derivatives of TEM and SHV enzymes having These all belong to the beta-lactamase function group 2be. The term has therefore been expanded to include: (i) enzymes with similar spectra to TEM and SHV mutants, but from other sources such as CTX-M type and VEB type , (Ii) TEM and SHV mutants with borderline ESBL activity, eg TEM-12, and (iii) various beta-lactamases that confer broader tolerance than the parent type but do not meet the definition of group 2be Eg, OXA derivatives, as well as mutant AmpC types with increased activity against cefepime.

  The terms “tolerant” and “tolerant”, as used herein, reduce or grow biological activity when a microorganism is exposed to antimicrobial concentrations that can be achieved with a normal therapeutic dosage regime in humans. Or the phenomenon which does not exhibit inhibition of proliferation. This means that infections caused by this microorganism cannot be successfully treated with this antibacterial agent.

  The term “microorganism” as used herein refers to a particularly pathogenic microorganism, such as, for example, a bacterium, yeast, fungus, intracellular or extracellular parasite. In preferred embodiments of the invention, the term refers to pathogenic or opportunistic bacteria. This includes both gram positive and gram negative bacteria. When referred to as gram-negative bacteria, it can be composed of bacteria from the following genera: Pseudomonas, Escherichia, Salmonella, Shigella, Enterobacter , Klebsiella, Serratia, Proteus, Campylobacter, Haemophilus, Morganella, Vibrio, Yersinia, Acinetobacter Genus (Acinetobacter), Blanchamella, Neisseria, Burkholderia, Citrobacter, Hafnia, Edwardiella, Aeromonas , Moraxella, Pasteurella, Providencia, Actinobacillus, Alcaligenes, Bordetella, Cedecea, Erwinia, Pantoea, Ralstonia, Stenotrophomonas, Xanthomonas and Legionella. When referred to as gram-positive bacteria, it can be composed of bacteria from the following genera: Enterococcus, Streptococcus, Staphylococcus, Bacillus, Listeria Clostridium, Gardnerella, Kocuria, Lactococcus, Leuconostoc, Micrococcus, Mycobacteria and Corynebacteria. When referred to as yeast and fungi, it can be composed of yeast from the following genera: Candida, Cryptococcus, Saccharomyces and Trichosporon.

  The term “mass spectrum” as used herein refers to a plot with molecular weight, or a function thereof (eg, mass to charge ratio (m / z), ion mass, etc.) as an independent variable. The dependent variable is typically a quantitative measurement, such as abundance, relative abundance, intensity, concentration, number of ions, number of molecules, number of atoms, number of counts / millivolt, number of counts, and the like. For example, for ions, the mass spectrum typically represents the mass-to-charge ratio (m / z) as an independent variable, where m is the mass of the ionic species, z is the charge of the ionic species, and the dependent variable is the most common Specifically, it is the abundance of each molecular ion or its fragment ion. The term “ion” means an atom or group of atoms that gains or loses one or more electrons, or gains or loses one or more protons, thereby gaining an overall charge. Ions can be formed in a variety of ways, such as destroying gas molecules by the effects of current, ultraviolet light and certain other light rays, high temperatures, and the like.

  The term “reference mass spectrum” as used herein refers to a control mass spectrum for comparative analysis.

  The term “modifying enzyme substrate compound” as used herein refers to any compound (whether or not an antibiotic) that can be hydrolyzed by an antibiotic modifying enzyme. This enzymatic modification of the substrate results in a reaction product with a different mass to charge ratio (or mass spectrum) than the original substrate compound. If the enzymatic modification is degradation, the reaction product may be referred to herein as a “degradation product”.

  The term “modifying enzyme” as used herein broadly refers to antibacterial compound modifying enzymes such as, for example, beta-lactamases.

  “Modification” as used herein refers to a chemical or physical (preferably chemical) modification that renders the compound inactive with respect to the antimicrobial activity of the antimicrobial compound. Modification can include degradation, which refers to the removal of chemical moieties from compound molecules resulting in lower molecular weights, optionally with modified mass to charge ratios. Alternatively, the modification can include a substitution or addition of a chemical moiety to the compound molecule that renders the compound inactive with respect to antibacterial activity, and this modified form results in a modified mass of the molecule With a modified mass to charge ratio.

  The term “cell lysate” as used herein refers to a cell suspension or fraction thereof obtained by disruption or lysis of cells. Unpurified cell lysates include all of proteins, glycoproteins, polysaccharides, lipids, and nucleic acids. In the embodiment of the present invention, the cell lysate may contain whole cells, but mainly consists of a cell portion obtained after the lysis step, an arbitrary fraction, or a mixture thereof. However, cell lysate solutions can include, but are not limited to, lysed cell solutions that have been treated to remove or inactivate selected molecules. This solution remains substantially “unpurified” for the most purified cell constituents. For example, the cell lysate can be a lysed cell solution treated with an agent that inactivates or removes the polymerase inhibitor. In addition, the cell lysate can be a lysed cell solution treated with an anticoagulant. Any method can be used to lyse the cells in the cell sample. For example, methods that can be used to lyse cells include osmotic shock, sonication, heating, physical disruption, microwave treatment, enzymes, and / or alkaline lysis.

  The term “medium” as used herein refers to a medium that contains all the elements necessary for the metabolic expression and / or growth of a microorganism. The medium can be solid, semi-solid, or liquid. Medium includes amino acids, peptones, carbohydrates, nucleotides, minerals, vitamins, active molecules (antibiotics, etc.), enzymes, surfactants, buffers, phosphates, ammonium salts, sodium salts, metal salts, enzyme activity A combination of one or more elements can be included, such as one or more substrates that allow detection.

  The term “supernatant” as used herein remains when cells grown in a liquid medium (eg, liquid broth) are removed by centrifugation, filtration, precipitation, or other methods well known in the art. Refers to liquid suspension, including lysates and suspensions.

  The terms “matrix material” and “MALDI matrix material”, as used herein, are interchangeable terms and may be used to form a matrix for use in MALDI mass spectra. Refers to the compound in it. In MALDI, it is necessary to embed the analysis object while there are an excessive amount of molecules that absorb the wavelength emitted by the laser. This matrix molecule is generally a small organic compound, mainly an acid. Suitable matrix materials for each type of laser used in MALDI are well known in the art, and the term “MALDI matrix material” is clearly understood by those skilled in the art. Without limiting the present invention, examples of commonly used matrix materials include sinapinic acid (SA), α-cyano-4-hydroxycinnamic acid (HCCA), 2.5-dihydroxybenzoic acid (DHB). 7-hydroxy-4- (trifluoromethyl) coumarin (HFMC), 3-hydroxypicolinic acid (3-HPA), 5- (trifluoro-methyl) uracil, caffeic acid, succinic acid, anthranilic acid, 3-amino Examples include pyrazine-2-carboxylic acid, tetrakis (pentafluorophenyl) porphyrine, and ferulic acid. The matrix is suitably dissolved in acetonitrile / water / formic acid (500: 500: 1, v / v / v) or at other suitable ratios depending on the matrix used.

  The term “sample”, as used herein, is a substance that contains or is suspected of containing an analyte, such as, for example, a characterized microorganism or beta-lactamase, or a beta-lactamase substrate, or a beta-lactamase degradation product. Point to. Samples useful in the methods of the present invention may be liquid or solid, may be dissolved or suspended in a liquid, may be in an emulsion or gel, and may be bound or absorbed by a substance. . The sample can be a biological sample, environmental sample, experimental sample, diagnostic sample, or other type of sample that is suspected of containing or containing the analyte of interest. Similarly, the sample can be or can include an organism, organ, tissue, cell, body fluid, biopsy sample, or fractions thereof. Samples useful in the methods of the invention may be any material suspected of containing analytes such as beta-lactamase and ESBL substrates. In the biological context, samples include biological fluids, whole organisms, organs, tissues, cells, microorganisms, culture supernatants, organelles, protein complexes, individual proteins, recombinant proteins, fusion proteins, viruses , Viral particles, peptides, and amino acids.

  The term “sample support” as used herein refers to any support suitable for receiving a sample for MALDI MS analysis. Commonly used is a 10 × 10 stainless steel target plate (Perseptive Biosystems (Flamingham, Mass., USA)), which can be hydrophobically coated if appropriate.

  The term “Michaelis-Menten constant”, often referred to as “Km”, as used herein refers to the substrate concentration at which the enzyme reaction rate is half the maximum rate. The term “maximum reaction rate” is often referred to as “Vmax” and, as used herein, refers to the maximum rate of an enzymatic reaction at a saturated substrate concentration. The Michaelis-Menten equation describes the production rate of molecules produced by enzymatic chemistry. To determine the maximum rate of the enzymatic reaction, the substrate concentration is increased until product production reaches a constant rate. This is the “maximum velocity” (Vmax) of the enzyme. In this state, the enzyme active site is saturated with the substrate. Since the substrate concentration at Vmax cannot be measured accurately, the enzyme can be characterized by the substrate concentration when the reaction rate is half the maximum rate. This substrate concentration is called the Michaelis-Menten constant (KM). For an enzyme reaction that exhibits a simple Michaelis-Menten equation, this represents the dissociation constant (substrate affinity) of the enzyme-substrate (ES) complex. The lower the value, the higher the affinity.

  The term “MALDI triple quadrupole MS” as used herein refers to a matrix-assisted laser desorption / ionization technique, where the mass spectrum is three quadrupoles arranged parallel to the incoming ions. Has poles. The first quadrupole functions as a mass filter. The second quadrupole acts as a collision cell, where the selected ions are broken into fragments. The resulting fragment is scanned by the third quadrupole. Quadrupole mass analyzers use an oscillating electric field to selectively stabilize or destabilize the trajectory of ions passing through a radio frequency (RF) quadrupole electric field. Only a single mass-to-charge ratio passes through the system from time to time, but with a magnetic lens potential change, quickly sweeps a wide range of m / z values, whether continuously or in a series of separate hops. Is possible. The quadrupole mass analyzer functions as a mass selection filter.

  The term “quantitative” as used herein refers to any method of obtaining a quantitative measurement. For example, quantification of a microorganism can include determining its abundance, relative abundance, intensity, concentration, count number, and the like.

  The term “structural biomolecule” as used herein refers to any protein, glycoprotein, polysaccharide, lipid, nucleic acid, etc., this amount being substantially between individual cells of a microbial culture. It can be used for quantification of microorganisms. For example, when DNA is used, quantification can be facilitated by DNA amplification or by the use of nucleic acid probes (identifiable by MS). Such a quantification method can use, for example, a standard calibration curve, where DNA content is plotted against cell number or another biomass parameter, such as the optical density or total carbon mass of the culture. To do.

  The term “metabolite” as used herein refers to a compound that is produced as a result of the function of a cell or organism's biochemical reaction, and this amount is substantially equal to the individual of a microbial culture. It is constant between cells and can be used for quantification of microorganisms.

Preferred Embodiments of the Invention The present invention provides a method for characterizing microbial antibiotic resistance. The first step in this method is to obtain one or more reference mass spectra of the antibiotic characterizing resistance, or a suitable pseudo-substrate thereof, or the molecular target of the antibiotic compound. The reference spectrum can be generated using any mass spectrum (MS) technique used to analyze the sample. A preferred MS technique is MALDI-MS.

  A suitable beta-lactamase substrate is any beta-lactam antibiotic. Alternatively, beta-lactam derivatives or mimetics that induce expression of microbial beta-lactamase or that are hydrolyzed by the enzymatic activity of beta-lactamase can be used. The pseudo-substrate used in the embodiments of the present invention may itself exhibit some antibiotic activity, but this is not necessary.

  Preferably, the beta-lactamase substrate is a compound in which the beta-lactamase degradation product is easily distinguishable by MS, preferably the substrate and the degradation product have different mass to charge ratios.

  A further step in a preferred method for characterizing a microorganism's resistance to beta-lactam antibiotics involves exposing the exposed sample to the substrate compound in aqueous solution by exposing the microorganism, its cell lysate, or its culture supernatant. To provide.

  A suitable exposed sample can be a body fluid or body tissue sample of a subject (ie, a human or animal subject) suspected of containing a microorganism that characterizes beta-lactam antibiotic resistance. A suitable body fluid sample may be a blood, stool or urine sample.

  Microbial exposure to substrate compounds can include in vivo and in vitro exposure.

  In certain embodiments, the exposure can include an incubation step, wherein the microorganisms are incubated in a solution containing the antimicrobial agent of interest for a short time, eg, 1-5 minutes and 1-3 hours. In addition, lysates of microorganisms and supernatants of microorganism cultures can also be used. When a particular enzyme is present, the antibacterial agent or its mimic substrate is modified or inactivated, resulting in a different component composition compared to the active drug form. Thereby, the mass of an antibacterial agent changes and it can detect by mass spectrometry.

  It is an advantage of the present invention that unpurified cell lysates can also be used as exposed samples. Thereby, the characterization microorganisms no longer need to be viable and there is no need to purify the exposed sample in order to be able to detect beta-lactamase activity.

  If a microorganism contains a beta-lactamase gene but does not produce the enzyme under normal growth conditions, the microorganism's beta-lactamase production is the cultivation of the microorganism in the presence of a beta-lactam antibiotic or a beta-lactamase-inducing compound. Can be triggered by. Preferably, a desired step of inducing or activating beta-lactamase production is performed prior to performing bacterial cell lysis.

  In general, the ability of an exposed sample to modify an antibiotic compound is either a decrease in the antibiotic substrate compound (or mimic thereof) or an increase in the reaction product of the hydrolysis reaction between the modifying enzyme and the substrate compound. Can be detected by detecting. Thus, beta-lactamase activity in an exposed sample can be detected by detecting either a decrease in beta-lactamase substrate compound or an increase in the reaction product of a hydrolysis reaction between beta-lactamase and substrate compound.

  Alternatively, the ability of an exposed sample to modify an antibiotic compound can be detected by detecting an alteration in the molecular target of the antibiotic compound. For example, resistance to erythromycin, ciprofloxacin, vancomycin, methicillin and tetracycline is based on targeted modifications such as RNA methylation. These target modifications can also be detected by mass spectrometry as described herein. Thus, the present invention is not limited to detecting beta-lactamase as a modified enzyme, ie, characterization of resistance to beta-lactam. Other resistances to antibiotic compounds that are not based on drug modifications can also be characterized using embodiments of the present invention. Beta-lactamases typically inactivate antibiotic drugs by hydrolysis, but other types of enzymatic modifications can be detected and characterized using the methods of the present invention. For example, aminoglycosides are modified by the addition of a phosphate moiety. Such substrate modification by the modifying enzyme can also be detected by the method of the present invention.

  It is an important discovery of the present invention that changes in reaction or target compounds (in quantitative amounts) can be measured very accurately by mass spectrometry. Thus, after the incubation step, exposed samples can be used for mass spectrometry using common mass spectrometry sample preparation protocols such as protein precipitation with organic solvents, solid phase extraction (SPE), and liquid-liquid extraction (LLE). Prepared. About 1 μL of the prepared solution is used for mass spectrometry. In a preferred embodiment of the invention, MALDI MS is used, more preferably MALDI quadrupole MS is used. By using MALDI MS, the exposure time (incubation time) can be made very short and the reaction compound can be measured with high accuracy. Characterization has been successfully achieved with an incubation time of about 5 minutes.

  Maldi MS involves applying an exposed sample together with a matrix material to a mass spectrometry sample support and drying the sample on the sample support to create a mass spectrometry sample. Suitable matrix materials are described herein above, and the nature of the matrix material is not particularly limited. Preparation of a mass spectrometry sample from an exposed sample can be carried out by methods well known to those skilled in the art of mass spectrometry.

  After mounting the sample on the mass spectrum, the mass spectrum of the sample is acquired by standard procedures according to the type of equipment used and the MS technique.

In the methods of the invention, detecting a substrate or target modification (eg, degradation or RNA methylation of a beta-lactamase substrate) is performed by MS, preferably by tandem mass spectrometry (MS-MS), or matrix-assisted laser dissociation / ionization. (MALDI). Mass spectrometry provides a powerful means for determining and identifying the structure of complex organic molecules, including proteins and peptides. In MS, high energy electrons collide with a sample compound and fragment in a specific form. The fragments have various weights and charges and pass through a magnetic field and separate according to their mass-to-charge ratio. The resulting specific pattern (mass spectrum) of the characteristic fragments of the sample compound is used to identify and quantify the compound. A typical MS procedure includes the following steps:
1. The sample is mounted on the MS device. If desired (in the case of a special form of MS called MALDI), the sample combined with the matrix is applied to a mass spectral sample support and the solvent or evaporation is allowed to dry the sample or mixture on the support.
2. The sample composition is ionized by one of a variety of methods (e.g., an electron beam impingement), thereby forming charged particles (ions).
3. The positive ions are accelerated by the electric field.
4). The mass-to-charge ratio (m / z) of the particle is calculated based on detailed information of the movement of the ion as it passes through the electromagnetic field.
5. Detect ions. The ions are classified according to m / z in step 4.

  In MALDI MS, the matrix consists of crystallized molecules, suitable examples of which include 3.5-dimethoxy-4-hydroxycinnamic acid (sinapic acid), α-cyano-4-hydroxycinnamic acid (alpha-cyano or alpha- Matrix), and 2.5-dihydroxybenzoic acid (DHB). The matrix solution is mixed with the exposed sample. Hydrophobic molecules are dissolved in the solution by the organic solvent, and water-soluble (hydrophilic) molecules are dissolved in the solution by the water. This solution is placed as a spot on a MALDI plate or support (usually a metal plate designed specifically for this purpose). The solvent evaporates, leaving a recrystallized matrix, which contains sample molecules dispersed throughout the matrix crystal.

  Suitable MS applications that can be used in aspects of the present invention include MALDI-TOF MS mass spectrometry, MALDI-FT mass spectrometry, MALDI-FT-ICR mass spectrometry, MALDI triple quadrupole mass spectrometry. Using MALDI-TOF mass spectrometry, throughput is estimated at 1 minute per sample. Using MALDI triple quadrupole mass spectrometry, the test time can be reduced to about 5 seconds per sample without loss of sensitivity or specificity.

  After acquiring the mass spectrum, the sample-derived mass spectrum can be qualitatively, semi-quantitatively or quantitatively compared with the reference mass spectrum of the antibacterial compound or its enzymatic modification product, molecular target, or modified enzyme substrate compound. Compare to. Such a comparison can determine the qualitative, semi-quantitative, or quantitative presence of the modification of the substrate or target, or the production of the modified product.

  Both inactivated or modified antibiotics (eg degradation products) and intact antibiotic substrates (or pseudo-substrates), as well as molecular targets can be measured simultaneously by mass spectrometry, eg, the substrate being examined is inactive The ratio of product to substrate can be used as an indicator of the ability of a microorganism to become or modify. Alternatively, or in addition, a decrease in the level of only the substrate in the sample, or an increase in the level of only the product in the sample is used as an indicator of the ability of the microorganism to inactivate or modify the antibiotic can do. Alternatively, increased levels of molecular targets, or increased levels of modified (resistant) targets can be used as an indicator of microbial resistance. In the case of characterization of antibiotic resistance to a drug that includes targeted modification as a resistance mechanism, the step of exposing a microorganism or its cell lysate or its culture supernatant to an antimicrobial compound in an aqueous solution is the It corresponds to the step of providing a sample of cell lysate, or its culture supernatant, and the step of detecting the modified target therein.

  Thus, in one embodiment of the aspect of the invention, the modification of the antimicrobial substrate compound is considered as evidence of, for example, the production of beta-lactamase by a microorganism, and the microorganism is, for example, by degradation or by an antimicrobial substrate compound or a suitable mimic. We show that it is likely to be resistant to beta-lactam antibiotic compounds inactivated by specific beta-lactamase when the substrate is applied. In this way, for example, the resistance of the microorganism to beta-lactam antibiotics can be characterized.

  Alternatively, in another embodiment of an aspect of the invention, the presence of a molecular target modification (relative amount or chemical composition) of an antimicrobial compound in a microbial cell indicates that the microorganism is an antibiotic of interest. Used as an indicator of high likelihood of resistance to the compound. In this way, the resistance of the microorganism to eg erythromycin, ciprofloxacin, vancomycin, methicillin and tetracycline can be characterized.

  In the foregoing aspects, the present invention provides, in certain embodiments, a method for rapidly diagnosing microorganisms that produce enzymes that inactivate or structurally modify antimicrobial agents. This method can be used for rapid detection of ESBL activity. Since third generation cephalosporins are widely used for the empirical treatment of patients with severe infections, this method is highly needed, especially in hospital settings. Rapid detection of ESBL activity in a patient sample is important in order to initiate the most appropriate antibiotic treatment for that patient as soon as possible. The method of the invention can be used for rapid detection of ESBL activity. Furthermore, the method of the present invention can be applied to microorganism supernatants and microorganisms directly isolated from patient samples after centrifugation of urine samples, for example. In this way, ESBL activity can be detected earlier than the detection of the (cultured) bacteria themselves.

  Mass spectrometry has not been used to detect enzymatic inactivation or chemical modification of antibiotics by monitoring a decrease in substrate strength or an increase in product strength. Furthermore, mass spectrometry has not been used to examine enzyme activity in complex samples such as lysed microorganisms. More specifically, the specific detection and characterization of ESBL enzymes by MS has never been reported before.

  Diagnostic methods for rapid detection of ESBL activity suitably include the release of beta-lactamase enzymes that inhibit antimicrobial agents from microorganisms by lysing the sample with a lysis reagent. This lysate is then transferred, preferably using an automatic pipetter, to a multi-well strip, such as an ATB® or Rapidec® strip, where the wells of the Contains reagents for performing various test reactions based on antibacterial compounds. Some wells contain a predetermined amount of one or more beta-lactamase substrates, for example in a dried or immobilized (glued) form. Other wells may contain one or more internal standards to facilitate quantification. Another well can also be used as a substrate-free control due to autolysis of the antimicrobial compound.

  After being transferred to a well and incubated in it for a short time as described herein, the exposed sample in each well can be suitably placed on a MALDI plate or other MS support. In the case of MALDI, a suitable matrix material is added to the support. Then, a mass spectrum is acquired. Spectral analysis is preferably performed using a dedicated analysis algorithm. The spectrum acquired from the exposed sample is compared to a reference spectrum using computer software to determine the presence of substrate degradation for each well. In the case of degradation, dedicated software can provide the test results as a report, which can include, for example: (1) microorganism identification (species name) (this identification can be simultaneous or (Obtained with parallel test strips, obtainable by reference test), (2) list of tested antimicrobial agents provided in multiwell test strips, (3) antimicrobial agents inhibited or degraded by the microorganism (4) Resistance mechanisms that appear to be the cause of antimicrobial inhibition, (5) Interpretation comments on each result.

  Alternatively, the step of exposing the microorganism, its cell lysate, and its culture supernatant to the aforementioned substrate compound in an aqueous solution (which provides an exposed sample) can be performed on a sample support for mass spectrometry. Can be done. A MALDI matrix can also be added directly to this exposed sample, if desired, when placing the beta-lactam enzyme on the sample support to degrade the substrate compound of the beta-lactamase enzyme.

  The methods of the invention can be performed with complex samples including unpurified cell lysates or patient samples. By this method, molecules can be accurately evaluated in the size range of antibacterial agents (usually 200 to 10,000 daltons). For example, the activity of antibiotic inactivating enzymes and antibiotic modifying enzymes using antibacterial agents as substrates. Can be suitably used to determine

  The method of the present invention can also be used as an ESBL confirmation test. In the majority of clinical microbial tests, bacteria are first screened for the ESBL phenotype, and then the ESBL phenotype is confirmed using another ESBL phenotype confirmation test. Aspects of the invention can be used to confirm bacterial ESBL phenotypes. Compared to current phenotyping, the inventive method proposed here has the potential advantage that it can be used not only in bacterial suspensions but also in bacterial lysates. The use of bacterial lysates can neutralize potential bias due to other mechanisms, particularly resistance based on reduced drug influx and increased efflux. Bacterial lysates cannot be combined with current phenotypic ESBL confirmation tests. This is because current phenotyping tests rely on bacterial growth.

  The methods of the present invention can also be used as a high-throughput screening procedure for new beta-lactamase inhibitors in the pharmaceutical industry. Currently, the beta-lactamase inhibitors clavulanic acid and tazobactam are used in combination with amoxicillin and piperacillin, respectively, to overcome the problems of beta-lactamase producing bacteria. Clavulanic acid or tazobactam inhibits beta-lactamase activity, while amoxicillin or piperacillin kills bacteria. The pharmaceutical industry considers the growing problem of ESBL and needs a screening tool for new compounds that inhibit ESBL. The present invention is very suitable for this purpose.

  We have used benzylpenicillin as a substrate, an unpurified lysate of E. coli producing CTX-M-1 and CTX-M-9, and K. pneumoniae producing SHV-2. ) Beta-lactamase activity was detected. We were able to monitor the enzyme dynamics of pure penicillinase (from B. cereus) using benzylpenicillin as a substrate.

Claims (12)

A method for characterizing microbial antibiotic resistance comprising:
(A) providing a reference mass spectrum of the molecular target of the antimicrobial compound compound;
(B) providing an exposed sample by exposing the microorganism to the antimicrobial compound in an aqueous liquid;
(C) obtaining a mass spectrum of the exposed sample;
(D) comparing the mass spectrum acquired in step (c) with the reference mass spectrum of step (a);
(E) after the exposure, it is determined from the comparison whether a modification of the chemical composition of the molecular target or an overproduction of the molecular target has occurred, and the modification or the overproduction is observed Confirming that the microorganism is potentially resistant to the antimicrobial compound;
Including a method.   The method of claim 1, wherein the antimicrobial compound is a beta-lactam antibiotic. After step (b), the exposed sample is combined with a matrix material and applied to a sample support for mass spectrometry,
The method of claim 1, wherein the sample is dried on the sample support to produce a sample for mass spectrometry for matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). 4. The method of claim 3 , wherein the mass spectrum is acquired using MALDI triple quadrupole mass spectrometry. In step (b), the microorganism is quantified in the sample by quantifying one or more structural biomolecules or metabolites derived from the microorganism;
The method of claim 1, wherein the structural biomolecule or the metabolite is a DNA molecule. 3. The method of claim 2 , wherein the antimicrobial compound is selected from the group comprising penicillin, cephalosporin, cephamycin, and carbapenem, or the group consisting of ceftazidime, cefotaxime, ceftriaxone, cefpodoxime, and aztreonam.   The method of claim 1, wherein the mass spectrum is acquired using one of MALDI triple quadrupole mass spectrometry, MALDI-TOF mass spectrometry, and MALDI-FTICR mass spectrometry. The method of claim 1, wherein the overproduction of the molecular target is detected by using an internal standard and observing an increased ratio of the molecular target to the internal standard. 9. The method of claim 8, wherein the internal standard is a nucleic acid. The method of claim 1, wherein the modification of the molecular target is methylation. The method of claim 10, wherein the molecular target is RNA. 12. The method of claim 11, wherein the antimicrobial compound is one of erythromycin, ciprofloxacin, vancomycin, methicillin, and tetracycline.