Method and apparatus for characterizing antibiotic resistance in microorganisms

Abstract

The present invention relates to a method for characterizing antibiotic resistance of a microorganism comprising the steps of: (a) providing a reference mass spectrum of an antimicrobial compound, an enzyme modification product thereof, a molecular target thereof or a substrate compound of a modifying enzyme thereof; (b) exposing a microorganism, its cell lysate, its growth medium supernatant to the antimicrobial compound or the substrate compound in aqueous liquid, thereby providing an exposed sample; (c) acquiring a mass spectrum of the exposed sample; (d) comparing the mass spectrum obtained in step c) with the reference mass spectrum obtained in step a), and (e) determining from said comparison whether a modification of the antimicrobial compound, its modification product, its molecular target or the substrate has occurred after said exposure, and establishing that the microorganism is potentially resistant to the antimicrobial compound when the modification is observed.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201080068659.0, filed on August 19, 2010, entitled "Method and apparatus for characterizing antibiotic resistance in microorganisms". TECHNICAL FIELD

[0002] The present invention relates to the field of bacterial diagnostics, in particular to a method for characterizing antibiotic resistance in microorganisms, a kit for carrying out the method of the invention and a system for characterizing antibiotic resistance in microorganisms, comprising a mass spectrometry apparatus for carrying out the method of the invention and sample preparation materials. BACKGROUND

[0003] Antibiotic resistance is the ability of a microorganism to resist the effects of an antibiotic. This resistance occurs through genetic mutation and plasmid exchange between microorganisms. Antibiotic resistance has had major impacts on medicine, and this impact will only grow in the coming years.

[0004] One group of opportunistic microorganisms that has regained interest due to antibiotic resistance is the Enterobacteriaceae. These species, including, for example, Klebsiella spp and Escherichia coli, include opportunistic pathogens that have been associated with urinary tract infections, septicemia, respiratory infections, and diarrhea, among others. It was known as early as three decades ago that these species were resistant to third generation cephalosporins, such as oximino β-lactams, but since then an exponential increase in resistance has been documented. Strains acquire their resistance through the production of enzymes called extended spectrum β-lactamases (ESBLs) that are able to inactivate third generation cephalosporins (ceftazidime, cefotaxime, ceftazidime) and monobactams (aztreonam). ESBLs are derivatives of common β-lactamases (e.g. TEM and SHV β-lactamases) that have undergone one or more amino acid substitutions near the active site of the enzyme, increasing its affinity and hydrolytic activity towards third generation cephalosporins and monobactams. The widespread use of new generation cephalosporins has prompted the evolution of more types of ESBLs. ESBLs are encoded by transmissible conjugative plasmids that are responsible for spreading resistance to other Gram-negative bacteria.

[0005] ESBLs are distinguished according to their physical characteristics into more than 450 varieties, and clavulanic acid, sulbactam and tazobactam inhibit ESBLs to varying degrees, a characteristic that can be used to detect ESBLs in the laboratory. Currently, only phenotypic ESBL detection tests are used in clinical microbiology laboratories. Molecular (genotypic) tests are under development. However, molecular tests have the problem of lacking 100% correlation between genotype and phenotype. Therefore, the predictive value of molecular tests of any bacterial phenotype, including ESBL-producing bacteria, is limited.

[0006] In general, current laboratory phenotypic tests are more sensitive and specific than ESBL genotypic confirmation tests. All phenotypic ESBL detection tests are based on the same principle: the test evaluates the change in the inhibitory effect on bacterial growth in the presence of a β-lactam antibiotic or a combination of a β-lactam antibiotic and a β-lactamase inhibitor. There are also various manual tests and automated platforms available from commercial sources that can be used to implement these phenotypic tests. Manual tests use discs or strips impregnated with a β-lactam antibiotic or a combination of a β-lactam antibiotic and a β-lactamase inhibitor. The impregnated material is placed on a solid culture medium previously inoculated with a bacterial suspension of known density. After overnight incubation, the inhibition of growth is determined by observation and quantified according to the diameter of the inhibition zone. Automated systems are based on the measurement of bacterial growth in the presence of different concentrations of a β-lactam antibiotic or a combination of a β-lactam antibiotic and a β-lactamase inhibitor. The results of such systems are obtained after 4 to 18 hours.

[0007] Currently, there is a need for devices and methods that can more rapidly diagnose ESBL-producing bacteria. There is also a need for ESBL detection tests that can be used in clinical microbiology laboratories in order to characterize ESBLs in terms of enzyme kinetics, so that evolutionary trends can be tracked and effective doses in antibiotic therapy can be evaluated and predicted. In general, these devices are widely used to characterize antibiotic resistance of microorganisms. SUMMARY

[0008] The present invention now provides devices and methods for rapidly diagnosing microorganisms that produce antibiotic-modifying enzymes, in particular (in preferred embodiments) ESBL-producing microorganisms. The invention also provides devices and methods that can characterize antibiotic-modifying enzymes themselves. Such characterization can detect new types of resistance early.

[0009] In a first aspect, the invention provides a method for characterizing antibiotic resistance of a microorganism, the method comprising the steps of:

[0010] a) providing a reference mass spectrum of an antimicrobial compound or of an enzymatic modification product thereof, of a molecular target thereof or of a modification enzyme substrate compound thereof;

[0011] b) exposing a microorganism, a cell lysate thereof or a growth medium supernatant thereof to said antimicrobial compound or to said substrate compound in an aqueous medium, thereby providing an exposed sample;

[0012] c) acquiring a mass spectrum of the exposed sample;

[0013] d) comparing the mass spectrum acquired in step c) with the reference mass spectrum in step a), and

[0014] e) determining from said comparison whether a modification of said antimicrobial compound, of a modification product thereof or of said substrate occurs after said exposure or whether a molecular target thereof is overproduced and establishing a potential resistance of said microorganism to said antimicrobial compound when said modification is observed.

[0015] In a preferred embodiment of the method, the modification comprises an enzymatic inactivation or enzymatic degradation of said antimicrobial compound and / or a methylation or overproduction of a molecular target thereof. More preferably, the enzymatic degradation is caused by a degradation by a beta-lactamase. In this case, the antimicrobial compound can be a beta-lactam antibiotic or any other beta-lactamase substrate. Thus, in another preferred embodiment of the method, a substrate compound for the enzyme modifying the antimicrobial compound can be used instead of the antimicrobial compound. In other preferred embodiments, the antimicrobial compound is a beta-lactam antibiotic, preferably selected from the group consisting of penicillins, cephalosporins, cephamycins and carbapenems, more preferably selected from the group consisting of ceftazidime, cefotaxime, ceftriaxone, cefpodoxime and aztreonam.

[0016] It is clear that, depending on the mechanism of antibiotic resistance, it is also possible that a molecular target of an antimicrobial compound (e.g. folate) is overproduced, which leads to resistance to a folate antagonist. Overproduction of a target can be detected by using an internal standard and observing an increase in the target / internal standard ratio. Nucleic acids (e.g. DNA) can be used as suitable internal standards.

[0017] Furthermore, again depending on the mechanism of antibiotic resistance, it is also possible to detect a methylation of a molecular target of an antimicrobial compound (e.g. like a nucleic acid).

[0018] In another preferred embodiment of the method of the application, the method can be performed by simultaneously exposing said microorganism to a plurality of antimicrobial compounds, thereby characterizing the resistance of said microorganism to a plurality of antibiotic compounds.

[0019] In another preferred embodiment of the method of the application, the enzyme inactivation or enzymatic degradation of the antimicrobial compound is caused by a beta-lactamase. In a particularly preferred embodiment, the beta-lactamase can be selected from the group consisting of cephalosporinases (including extended-spectrum cephalosporinases), penicillinases, carbenicillinases, cloxacillinases and carbapenamases.

[0020] In a further preferred embodiment of the method of the application, the beta-lactamase is an extended-spectrum beta-lactamase (ESBL).

[0021] In a further preferred embodiment of the method, the microorganism is a microorganism that can produce an ESBL, preferably a Gram-negative bacterium selected from the group consisting of Klebsiella pneumoniae, Escherichia coli, Klebsiella oxytoca and Proteus mirabilis.

[0022] The sample used in the aspects of the application comprises a microorganism or a lysate thereof. The microorganism sample can be a microorganism culture sample. Such cultures do not have to be pure cultures. Alternatively, parts of the culture medium or direct clinical material can also serve as sample source.

[0023] In another preferred embodiment of the method, the method is also part of a method for characterizing an antimicrobial-modifying enzyme of a microorganism, preferably the antimicrobial-modifying enzyme is an extended-spectrum beta-lactamase (ESBL). In a particularly preferred embodiment, the method of the application is part of a method for characterizing an extended-spectrum beta-lactamase (ESBL).

[0024] Preferably, the method for characterizing the enzyme according to the application comprises determining the rate of modification (preferably degradation) of the antimicrobial compound or the substrate compound and / or the rate of production of the enzymatic modification product of the compound or substrate or the overproduction of the compound molecular target in the presence or absence of a specific enzyme inhibitor, thereby determining the Michaelis-Menten (Km) constant and the maximum reaction rate of the enzyme.

[0025] In another preferred embodiment of the method, the mass spectrum is acquired using MALDI triple quadrupole mass spectrometry.

[0026] In another preferred embodiment of the method, the exposed sample is a crude cell lysate of the exposed microorganism.

[0027] In another preferred embodiment of the method, the method further comprises the step of quantitatively analyzing the microorganism. Preferably, the microorganism is quantitatively analyzed by quantitatively analyzing one or more structural biomolecules or metabolites derived from the microorganism in the sample. In a preferred embodiment, the structural biomolecule or metabolite is selected from nucleic acids, preferably (genomic) DNA. DNA is present in cells as single molecules, which can be quantitatively analyzed by using, for example, PCR and / or DNA probe-mediated techniques.

[0028] In another aspect, the present application provides a kit for characterizing a microorganism for beta-lactam antibiotic resistance, comprising:

[0029] a) a lysis buffer for lysing the microorganism;

[0030] b) at least one antimicrobial compound or a substrate for an antimicrobial compound modifying enzyme, and

[0031] c) a MALDI matrix,

[0032] Preferably the kit further comprises:

[0033] d) a carrier carrying at least one antimicrobial compound or substrate, wherein the carrier is optionally in the form of a disposable mass spectrometry sample carrier plate.

[0034] In another aspect, the present application provides a system suitable for characterizing a microorganism for beta-lactam antibiotic resistance by the above-mentioned method of the present application, the system comprising one or more of the following components:

[0035] - at least one antimicrobial compound or a substrate for an antimicrobial compound modifying enzyme;

[0036] - a container for exposing the microorganism, its cell lysate or its growth medium supernatant to the at least one antimicrobial compound in the aqueous liquid, preferably the at least one substrate compound is provided in the container;

[0037] - a lysis buffer for lysing the microorganism species;

[0038] - a MALDI matrix;

[0039] - a mass spectrometry device;

[0040] - a reference mass spectrum of the antimicrobial compound, its enzymatically modified product, its molecular target or its modifying enzyme substrate compound, and

[0041] - a mass spectrometry sample carrier,

[0042] optionally further comprising

[0043] - an automated pipettor for liquid handling;

[0044] - a computer program comprising computer program code means which, when said program is run on a computer, implements all the steps of the method of the present application as described above, including for example result translation algorithms, interface software and / or expert system software.

[0045] The present application provides in another aspect a computer program comprising computer program code means which, when said program is run on a computer, implements all the steps of the method of the present application as described above.

[0046] In another aspect, the present application provides a computer program product comprising computer program code means stored on a computer readable medium which, when said program product is run on a computer, implements the method of the present application. DETAILED DESCRIPTION

[0047] Definitions

[0048] The terms "antibiotic" and "antimicrobial compound" are used interchangeably herein and are used herein to describe a compound or composition that reduces the viability of or inhibits the growth or reproduction of a microorganism. By "inhibits the growth or reproduction" is meant that the generation time is increased at least 2-fold, preferably at least 10-fold, more preferably at least 100-fold, and most preferably indefinitely, that is, all cells die. As used in this disclosure, antibiotic is also intended to include an antibacterial, bacteriostatic, or bactericidal agent. Non-limiting examples of antibiotics useful in the present application include penicillins, cephalosporins, aminoglycosides, sulfonamides, macrolides, tetracyclines, lincosamides, quinolones, chloramphenicol, glycopeptides, metronidazole, rifampin, isoniazid, spectinomycin, folate inhibitors, sulfamethoxazole, and the like.

[0049] The term "β-lactam antibiotic" is used to refer to a compound having antibiotic properties including β-lactam efficacy. A β-lactam ring (β-lactam) is a cyclic amide containing a heterocyclic structure composed of three carbon atoms and one nitrogen atom. Non-limiting examples of β-lactam antibiotics useful in the present application include penicillins, cephalosporins, cephamycins, penems, carbapenems, and monobactams. β-lactam antibiotics are effective against a variety of bacterial infections (in the absence of antibiotic resistance). The term "β-lactam antibiotic" as used herein refers to any antibiotic that has undergone a qualitative or structural change upon inactivation by an antibiotic resistant microorganism, if the qualitative or structural change is detectable by mass spectrometry.

[0050] The term "third generation cephalosporin" refers to a class of compounds including, but not limited to, cefixime, ceftazidime, cefotaxime, ceftriaxone, cefcapene, cefdaloxime, cefdinir, cefditoren, cefetamet, cefmenoxime, cefoperazone, cefotetan, cefpodoxime, ceftalozane, ceftobiprole, ceftizoxime, ceftominazole, ceftobiprole, cefpodoxime, ceftalozane, ceftobiprole, ceftazidime, and oxacephem.

[0051] The term "beta-lactamase" refers to an enzyme (EC 3.5.2.6) produced by microorganisms, preferably bacteria, which is capable of hydrolyzing the beta-lactam ring of beta-lactam class antibiotics. This class of enzymes is generally divided into four major groups (Ambler classes A, B, C, and D) based primarily on protein homology according to the so-called Ambler classification. Examples of beta-lactamases include cephalosporinases, penicillinases, carbeniciUinases, cloxacillinases, carbapenemases, and ceftazidimeases. This term includes "classical" beta-lactamases, extended-spectrum beta-lactamases (ESBLs), and AmpC beta-lactamases. Preferred beta-lactamases in the present application are beta-lactamases of Ambler classes A and D, or according to Bush classification, beta-lactamases of class 2 (Bush et al. 1995. Antimicrob Agents Chemother. 39:1211-33). Class A antibiotics in the Ambler classification are typical serine beta-lactamases of the active site, while class D antibiotics in the Ambler classification are a specific group of serine beta-lactamases that have little sequence similarity to class A beta-lactamases, and are colloquially known as OXA (oxacillinase) class. Metallo-carbapenemases are also preferred.

[0052] The term "extended-spectrum beta-lactamase" (abbreviated ESBL) was originally termed "extended-spectrum beta-lactamase" and was first invented for derivatives of TEM and SHV enzymes, which are capable of hydrolyzing oximino cephalosporins. These belong to the beta-lactamase functional class 2be. The term has since been expanded to include: (1) enzymes similar in spectrum to TEM and SHV mutants but derived from other sources, such as the CTX-M and VEB types; (2) TEM and SHV mutants with borderline ESBL activity, such as TEM-12; and (3) various beta-lactamases that provide a broader resistance than their parent types but do not fit the definition of class 2be, such as OXA derivatives with enhanced activity against cefepime and mutants of the AmpC type.

[0053] The terms "resistant" and "resistance" as used herein refer to the phenomenon that the viability of a microorganism is not reduced and growth or reproduction is not inhibited when the microorganism is exposed to a concentration of an antimicrobial agent that can be reached by a normal human treatment dosage regimen. This means that an infection caused by such a microorganism cannot be treated successfully using such an antimicrobial agent.

[0054] The term "microorganism" as used herein refers especially to pathogenic microorganisms, such as bacteria, yeasts, fungi and intracellular and extracellular parasites. In a preferred aspect of the application, the term refers to pathogenic bacteria or opportunistic bacteria. These include Gram-positive and Gram-negative bacteria. Gram-negative bacteria can be bacteria of the genera Pseudomonas, Escherichia, Salmonella, Shigella, Enterobacter, Klebsiella, Serratia, Proteus, Campylobacter, Haemophilus, Morganella, Vibrio, Yersinia, Acinetobacter, Branhamella, Neisseria, Burkholderia, Citrobacter, Hafnia, Edwardsiella, Aeromonas, Moraxella, Pasteurella, Providencia, Actinobacillus, Alcaligenes, Bordetella, Cedecea, Erwinia, Pantoea, Ralstonia, Stenotrophomonas, Xanthomonas and Legionella. Gram-positive bacteria can be bacteria of the genera Enterococcus, Streptococcus, Staphylococcus, Bacillus, Listeria, Clostridium, Gardnerella, Kocuria, Lactococcus, Micrococcus, Leuconostoc, Mycobacteria and Corynebacteria.Yeast and fungi can be yeast of the genera Candida, Cryptococcus, Saccharomyces, and Trichosporon.

[0055] The term "mass spectrum" as used herein refers to a plot with molecular mass or a function thereof (e.g., mass-to-charge ratio (m / z), ion mass, etc.) as the independent variable. The dependent variable is typically a quantitative measure, such as abundance, relative abundance, intensity, concentration, ion number, molecule number, atom number, counts / mV, counts, etc. For example, in an ion environment, mass spectra typically have mass-to-charge ratio (m / z) as the independent variable, where m is ion mass and z is ion charge, and the dependent variable most often is the abundance of each molecular ion and / or its fragment ions. Ions can be formed in a variety of ways, including the dissociation of gas molecules under the influence of electric currents, ultraviolet and certain other rays, and / or high temperatures.

[0056] The term "reference mass spectrum" as used herein refers to a control mass spectrum for comparative analysis.

[0057] The term "substrate compound of a modifying enzyme" as used herein refers to any compound (whether or not an antibiotic) that can be hydrolyzed by an antibiotic-modifying enzyme. Enzymatic modification of the substrate results in a reaction product having a different mass-to-charge ratio (or mass spectrum) than the original substrate compound. As used herein, "reaction product" refers to "degradation product" if the enzymatic conversion is enzymatic degradation.

[0058] The term "modifying enzyme" as used herein refers broadly to an antimicrobial compound-modifying enzyme, such as a β-lactamase.

[0059] "Modification" as used herein refers to a chemical or physical change (preferably a chemical change) in an antimicrobial compound that inactivates the antimicrobial activity of the compound. Modification can include degradation, which refers to the loss of a chemical group from a compound molecule, resulting in a decrease in molecular mass, sometimes accompanied by a change in mass-to-charge ratio. Alternatively, modification refers to the replacement or addition of a chemical group to a compound molecule, resulting in a change in mass of the molecule, sometimes accompanied by a change in mass-to-charge ratio, that inactivates the antimicrobial activity of the compound.

[0060] As used herein, the term "cell lysate" refers to a suspension of cells or cell fragments obtained by disrupting or lysing cells. A crude cell lysate contains all proteins, glycoproteins, polysaccharides, lipids, and nucleic acids. The cell lysate of the present application can include whole cells, but is essentially composed of cellular components or any fragments obtained after the lysing step or mixtures thereof. However, the cell lysate solution can include, but is not limited to, a lysed cell solution that can be treated to remove or inactivate selected molecules. Thus, the solution is still essentially "crude" as compared to the most purified cellular components. For example, the cell lysate can be a lysed cell solution that has been treated with a reagent that inactivates or removes polymerase inhibitors. In addition, the cell lysate can be a lysed cell solution that has been treated with an anticoagulant. Any method can be used to lyse cells in a cell sample. For example, osmotic shock, sonication, heat, physical disruption, microwave treatment, and enzymatic and / or alkaline lysis are methods that can be used to lyse cells.

[0061] As used herein, the term "growth medium" refers to a medium that contains all elements required for the expression of genes by a microorganism and / or the growth of a species of microorganism. The growth medium can be a solid medium, a semi-solid medium, or a liquid medium. The growth medium can contain one or more elements, such as amino acids, peptones, carbohydrates, nucleotides, minerals, vitamins, active molecules such as antibiotics, enzymes, surfactants, buffers, phosphates, ammonium salts, sodium salts, metal salts, one or more substrates that can detect the activity of an enzyme, and the like.

[0062] As used herein, the term "supernatant" refers to the liquid suspension remaining after cells grown in a liquid medium, such as a liquid broth medium, are removed by centrifugation, filtration, sedimentation, or other known methods, which also contains dissolved and suspended material.

[0063] The terms "matrix" and "MALDI matrix" are used interchangeably herein and refer to a liquid or solid compound that can be used to form a matrix for MALDI mass spectrometry. In order to perform MALDI, the analyte must be embedded in a large number of molecules that have good absorption characteristics at the wavelength of the laser used. These matrix molecules are usually small molecule organic compounds, primarily acids. Suitable matrix materials for each laser used in MALDI are well known in the art and will be readily apparent to one skilled in the art upon reading the present disclosure. Without limiting the present application, commonly used matrix materials include sinapinic acid (SA), alpha-cyano-4-hydroxycinnamic acid (HCCA), 2,5-dihydroxybenzoic acid (DHB), 7-hydroxy-4-(trifluoromethyl)coumarin (HFMC), 3-hydroxypyridine carboxylic acid (3-HPA), 5-(trifluoromethyl)uracil, caffeic acid, succinic acid, anthranilic acid, 3-aminopyrazine-2-carboxylic acid, tetra(pentafluorophenyl)porphyrin, and ferulic acid. The matrix is suitably dissolved in acetonitrile / water / formic acid (500:500:1; v / v / v), or other suitable ratios depending on the matrix used.

[0064] The term "sample" as used herein refers to a substance containing or suspected of containing an analyte, such as a microorganism or β-lactamase to be characterized, or a β-lactamase substrate or β-lactamase degradation product thereof. The sample used in the methods of the present application can be in a liquid or solid state, soluble or suspended in a liquid, emulsion or gel, or bound to or adsorbed by a substance. The sample can be a biological sample, environmental sample, experimental sample, diagnostic sample, or any other type of sample containing or suspected of containing an analyte of interest. Likewise, the sample can be or can comprise an organism, organ, tissue, cell, bodily fluid, biopsy sample, or fragment thereof. The sample used in the methods of the present application can be any substance suspected of containing an analyte, such as a β-lactamase and substrate for ESBLs. In a biological context, the sample can comprise a biological fluid, whole organism, organ, tissue, cell, microorganism, culture supernatant, subcellular organelle, protein complex, monomeric protein, recombinant protein, fusion protein, virus, virion, peptide, and amino acid.

[0065] The term "sample carrier" as used herein refers to any carrier suitable for receiving a sample for MALDI analysis. A commonly used carrier is a 10 x 10 stainless steel target plate (Perseptive Biosystems, Framingham, MA, USA), which can be coated with a hydrophobic coating if appropriate.

[0066] The term "Michaelis-Menten constant" or "Km" as used herein refers to the substrate concentration at which the rate of an enzyme reaction is at one-half of its maximum value. The term "maximum reaction rate" or "Vmax" as used herein refers to the maximum rate of an enzyme reaction at saturating substrate concentrations. Michaelis-Menten kinetics describes the rate of production of molecules from an enzyme chemical reaction. To determine the maximum rate of an enzyme reaction, the substrate concentration should be increased until the rate of product formation is constant. This is the "maximum rate" (Vmax) of the enzyme. Since the substrate concentration cannot be accurately measured at Vmax, the enzyme can be characterized by the substrate concentration at which the maximum rate is one-half. This substrate concentration is the Michaelis-Menten constant (KM). For enzyme reactions that exhibit simple Michaelis-Menten kinetics, this represents the dissociation constant of the enzyme-substrate (ES) complex (the affinity of the substrate). The lower the value, the higher the affinity.

[0067] The term "MALDI triple quadrupole MS" as used herein refers to a method of matrix-assisted laser desorption / ionization in which the mass spectrometer has three quadrupoles in line with the incoming ions. The first quadrupole acts as a mass filter. The second quadrupole acts as a collision cell in which selected ions fragment into fragments. The third quadrupole scans the resulting ion fragments. Quadrupole rod mass analyzers use oscillating electric fields to selectively stabilize or destabilize the trajectory of ions passing through a radio frequency (RF) quadrupole field. Only one mass-to-charge ratio can pass through the system at any time, while changing the electric potential on the magnetic lens can let multiple m / z values swing rapidly, in a sustained fashion or in a continuous discrete hop fashion. The quadrupole rod mass analyzer acts as a mass-selective filter.

[0068] The term "quantitative analysis" as used herein refers to any method used to obtain a quantitative measurement. For example, quantitative analysis of microorganisms includes determining the abundance, relative abundance, intensity, concentration, and / or count of microorganisms, among others.

[0069] The term "structural biomolecule" as used herein refers to any cellular protein, glycoprotein, polysaccharide, lipid, nucleic acid, etc., the quantity of which is substantially constant from cell to cell in a microbial culture and which can be used to quantitatively analyze those microorganisms. For example, if DNA is used, then quantitative analysis can be performed by DNA amplification or using nucleic acid probes that can be identified by mass spectrometry. Such quantitative analysis methods can employ, for example, standard calibration curves in which a plot of DNA content versus cell number or other biomass parameter (e.g., culture optical density or total mass of carbon) can be made.

[0070] The term "metabolite" as used herein refers to a compound produced by biochemical reactions in a cell or organism, the quantity of which is substantially constant from cell to cell in a microbial culture and which can be used to quantitatively analyze those microorganisms.

[0071] Preferred embodiments

[0072] The present invention provides a method for characterizing antibiotic resistance of a microorganism. The first step of the method is to provide one or more reference mass spectrometric characterizations of the antibiotic compound to be characterized for resistance thereto, a suitable surrogate substrate for the antibiotic compound, and a molecular target of the antibiotic compound. The reference spectra can be made by using any mass spectrometric (MS) technique for sample analysis. Preferably, the MS technique is MALDI-MS.

[0073] The β-lactamase substrate used can be any β-lactam antibiotic. Alternatively, a β-lactam derivative or surrogate can be used that induces expression of β-lactamase in the microorganism and / or is capable of being hydrolyzed by the enzymatic activity of β-lactamase. The surrogate substrate used in the present invention can, but need not, exhibit any antibiotic activity per se.

[0074] The β-lactamase substrate is preferably a compound that is readily identifiable by MS, preferably the substrate and its degradation product have different mass-to-charge ratios.

[0075] Another step of the preferred method for characterizing β-lactam antibiotic resistance of a microorganism involves exposing the microorganism, a cell lysate thereof, or a supernatant of a growth medium thereof to the substrate compound in an aqueous solution, thereby providing an exposed sample.

[0076] The exposed sample can be a body fluid or body tissue sample of a subject (i.e., a human or animal subject) suspected of carrying the microorganism to be characterized for β-lactam antibiotic resistance. The body fluid sample can be a blood, fecal, or urine sample.

[0077] The microorganism can be exposed to the substrate compound in vivo or in vitro.

[0078] In certain embodiments, the exposure can comprise a culturing step, wherein the microorganism is cultured for a short period of time, e.g., 1-5 minutes to 1-3 hours in a solution containing the antimicrobial agent of interest. In addition, lysates of the microorganism and supernatants of the microorganism medium can also be used. In the presence of the specific enzyme, the antimicrobial agent or its surrogate substrate is modified or inactivated, thereby resulting in a difference in elemental composition as compared to the active drug form. This results in a change in the mass of the antimicrobial agent that can be detected by mass spectrometry.

[0079] An advantage of the present invention is that crude cell lysates can also be used to provide the exposed sample. Thus, the microorganism to be characterized need not be viable, and the exposed sample need not be purified prior to detecting β-lactamase activity therein.

[0080] When the microorganism contains a beta-lactamase gene but does not produce the enzyme under the prevailing growth conditions, the microorganism can be induced to produce beta-lactamase in the organism by culturing the microorganism in the presence of a beta-lactam antibiotic or a beta-lactamase inducing compound. Preferably, the production of beta-lactamase can be induced or stimulated selectively prior to lysis of the bacterial cells.

[0081] In general, the ability of the exposed sample to modify the antibiotic compound can be detected by detecting a decrease in the amount of antibiotic substrate compound, or by detecting an increase in the amount of reaction product of the hydrolysis reaction between the modifying enzyme and the substrate compound. Thus, the activity of beta-lactamase in the exposed sample can be detected by detecting a decrease in the amount of beta-lactamase substrate compound, or by detecting an increase in the amount of reaction product of the hydrolysis reaction between the beta-lactamase and the substrate compound.

[0082] Alternatively, the ability of the exposed sample to modify the antibiotic compound can also be detected by detecting a modification of the molecular target of the antibiotic compound. For example, resistance to erythromycin, ciprofloxacin, vancomycin, methicillin and tetracycline is based on target modification, such as RNA methylation. Again, these target modifications can be detected by mass spectrometry as described herein. Thus, the present application is not limited to detecting beta-lactamase as the modifying enzyme, but can also be used to characterize resistance to beta-lactams. Again, other resistances to antibiotics that are not based on drug modification can also be characterized using the present application. Although beta-lactams typically inactivate antibiotic drugs by hydrolysis, other types of enzyme modifications can also be detected and characterized using the apparatus and methods of the present application. For example, aminoglycoside antibiotics can be modified by the addition of phosphate groups. Such modification of the modifying enzyme substrate can also be detected by the methods of the present application.

[0083] It is an important finding of the present inventors that the change in reaction or target compound can be measured (quantified) very precisely by mass spectrometry. Thus, after the culturing step, the exposed sample can be prepared for mass spectrometry using general sample preparation methods, such as by protein precipitation with organic solvents, solid phase extraction (SPE) or liquid-liquid extraction (LLE). About 1 µL of the prepared solution is used for mass spectrometry. In a preferred embodiment of the present application, MALDI MS is used, more preferably MALDI quadrupole MS. Using MALDI MS, the reaction compound can be measured precisely, and the exposure time (incubation period) can be very short. Successful characterization is possible with incubation times of about 5 minutes.

[0084] MALDI MS involves coating the exposed sample, together with a matrix, onto a mass spectrometry sample plate and drying the sample on the sample plate to prepare a mass spectrometry sample. Suitable matrices are detailed above, and the nature of the matrix is not particularly limited. The mass spectrometry sample can be prepared from the exposed sample by methods known to those skilled in the art of mass spectrometry.

[0085] After the sample has been placed on the mass spectrometer, the mass spectrum of the sample can be acquired by standard procedures, which depend on the equipment used and the type of MS method.

[0086] In the methods of the application, MS, preferably tandem mass spectrometry (MS-MS) or matrix assisted laser desorption / ionization (MALDI) is employed to carry out the substrate or target modification (e.g. beta-lactamase substrate degradation or RNA methylation) step. Mass spectrometry provides an effective method for determining the structure and identity of complex organic molecules, including proteins and peptides. In MS, sample compounds are bombarded with high energy electrons, causing them to fragment in a characteristic way. These fragments, which vary in mass and charge, are then passed through a magnetic field and separated according to their mass-to-charge ratio. The final characteristic fragmentation pattern (mass spectrum) of a sample compound can be used to identify and quantitatively analyze that compound. A typical MS procedure involves the following steps:

[0087] 1. The sample is loaded into the MS instrument by coating the sample, optionally (in the case of a special form of MS known as MALDI) together with a matrix, onto a mass spectrometry sample plate, and drying the sample or mixture on the plate by evaporation of the solvent.

[0088] 2. The sample components are ionized by one of a variety of methods (e.g. by bombarding the sample with an electron beam), thereby forming charged particles (ions)

[0089] 3. The positive ions are accelerated by an electric field

[0090] 4. The mass-to-charge ratio (m / z) of the particles is calculated from the detailed information on the motion of the ions in the electromagnetic field, and

[0091] 5. The ions arranged according to m / z in step 4 are detected.

[0092] In MALDI MS, the matrix consists of crystalline molecules, of which three suitable crystalline molecules are exemplified by 3,5-dimethoxy-4-hydroxycinnamic acid (sinapinic acid), a-cyano-4-hydroxycinnamic acid (a-cyano or a-matrix), and 2,5-dihydroxybenzoic acid (DHB). The matrix solution is mixed together with the exposed sample. Organic solvents solubilize hydrophobic molecules in solution, while water solubilizes water-soluble (hydrophilic) molecules in solution. The solution is spotted on a MALDI plate or substrate (usually a specially prepared metal plate). The solvent is evaporated, leaving behind the recrystallized matrix and the sample molecules dispersed on the matrix crystals.

[0093] Suitable MS applications that can be used in the present application include MALDI-TOF MS mass spectrometry, MALDI-FT mass spectrometry, MALDI-FT-ICR mass spectrometry, MALDI triple quadrupole mass spectrometry. With MALDI-TOF mass spectrometry, the throughput is estimated to be 1 minute per sample. With MALDI triple quadrupole mass spectrometry, the duration of the assay is reduced to about 5 seconds per sample without loss of sensitivity or specificity.

[0094] After acquisition of the mass spectrum, the derived sample mass spectrum is compared to reference mass spectra of the antimicrobial compound, of its enzymatic modification products, of its molecular target, and also to reference mass spectra of the substrate compound of its modifying enzyme, in a qualitative, semi-quantitative or quantitative analysis. Through such comparison, the modification of the substrate or target and / or the production of the modification product can be determined in a qualitative, semi-quantitative or quantitative analysis.

[0095] The mass spectrometry analysis can measure both the inactivated or modified antibiotic (e.g. degradation product) and the unimpaired antibiotic substrate (or mimic substrate) and the molecular target, and the ratio of product to substrate can measure the ability of the microorganism to inactivate or modify the measured substrate. Alternatively or in addition, either a decrease in substrate levels or an increase in product levels in the sample can be a measure of the ability of the microorganism to inactivate or modify the antibiotic. Alternatively, an increase in the level of the molecular target or an increase in the level of the modified (resistant) target can be indicative of resistance of the microorganism. In the case of characterizing antibiotic resistance to a drug involving modification of the target as a resistance mechanism, the step of exposing the microorganism, its cell lysate or its growth medium supernatant to the antimicrobial compound in an aqueous solution is equivalent to providing a sample of the microorganism, cell lysate or growth medium supernatant and detecting the modified target therein.

[0096] Thus, in one embodiment of the application, the modification of the antimicrobial substrate compound is evidence of the production of a β-lactamase enzyme by the microorganism and indicates that the microorganism can be resistant to, for example, a β-lactam antibiotic compound provided by degradation, inactivation by the specific β-lactamase enzyme of the antimicrobial substrate compound or applicable surrogate substrate. For example, by this method, the resistance of a microorganism species to a β-lactam antibiotic can be characterized.

[0097] Alternatively, in another embodiment of the application, the modification (relative amount or chemical modification) of the antimicrobial compound molecular target in the microorganism cell indicates that the microorganism can be resistant to the target antibiotic compound. For example, by this method, the resistance of a microorganism species to erythromycin, ciprofloxacin, vancomycin, methicillin and tetracycline can be characterized.

[0098] As described above, the present application provides, in certain embodiments, a method for rapid diagnosis of microorganisms that produce enzymes that inactivate or structurally modify antimicrobial agents. This method can be used to rapidly detect ESBL activity. This method is extremely desirable, particularly in hospitals, because third generation cephalosporins are widely used for empirical therapy in critically ill patients for infections. Rapid detection of ESBL activity in patient samples is extremely important for the earliest possible start of the most appropriate antibiotic drug therapy for the patient. The method in the present application can be used to rapidly detect ESBL activity. Furthermore, the method in the present application is equally applicable to microorganism culture supernatants or directly from microorganisms isolated from patient samples such as urine samples after centrifugation. By this method, it should be possible to detect ESBL activity even before the (cultured) bacteria are detected.

[0099] Mass spectrometry has not been used to detect enzymatic inactivation or chemical modification of antibiotics by monitoring the decrease in substrate intensity and / or the increase in product intensity. Furthermore, mass spectrometry has not been used to study enzyme activity in complex samples (e.g., lysed microorganisms). More specifically, ESBL enzyme detection and characterization using MS has never been published before.

[0100] A diagnostic method for rapid detection of ESBL activity also requires the appropriate release of the β-lactamase enzyme that inhibits antimicrobial agents from the microorganism by lysing the sample using a lysing reagent. Subsequently, these lysates can be transferred, preferably using an automated pipettor, to a multi-well test strip, such as the ATB TM or Rapidec TMThe test strips, which contain a number of wells with reagents, allow for different test reactions depending on the different antimicrobial compounds. For example, some wells contain a certain amount of one or more β-lactamase substrates in dry or immobilized (glued) form. Some wells can also contain one or more internal standards to facilitate quantitative analysis. Some wells can also be left empty of substrate as a control for self-degradation of the antimicrobial compound.

[0101] After transfer into the wells and incubation for a short period of time as described herein, the exposed sample of each well is suitable for placement on a MALDI plate or other MS carrier plate. In the case of MALDI, the applicable matrix is added to the carrier plate. Thereafter, the mass spectrum can be acquired. Suitable mass spectral analysis is performed using specialized analysis algorithms. Subsequently, the mass spectrum obtained from the exposed sample is compared to the reference mass spectrum using computer software in order to determine whether degradation of the substrate has occurred in each well. If degradation has occurred, the specialized software can provide the test results in the form of a report, which can contain such things as: (1) the species (name) of the microorganism (identification of the microorganism species can be performed by reference testing, also in the same or parallel test strips), (2) a list of the antimicrobial agents tested provided in the multi-well test strip, (3) a list of the antimicrobial agents inhibited or degraded by the microorganism species, (4) a hypothesis of the resistance mechanism responsible for the antimicrobial inhibition, (5) specialized explanatory notes on the results.

[0102] Alternatively, the step of exposing the substrate compounds to the aqueous liquid in the presence of the microorganism, its cell lysate or its growth medium supernatant (thereby providing the exposed sample) can be performed on a mass spectral carrier plate. The MALDI matrix can be added directly to the exposed sample while allowing the β-lactamase, if present, to degrade the β-lactamase substrate compounds on the sample carrier plate.

[0103] The method in the present application can be performed using complex samples, including crude cell lysates or patient samples. The method allows for the precise evaluation of molecules in the size range of antimicrobial agents (typically 200 to 1000 Daltons) and can be suitable for determining the activity of antibiotic inactivation or antibiotic modifying enzymes using, for example, antimicrobial drugs as substrates.

[0104] The method of the present invention can also be used as an ESBL confirmatory test. In most clinical microbiology laboratories, bacteria are first screened for the ESBL phenotype, and then a separate ESBL phenotype confirmatory test can be used to determine the ESBL phenotype. The present invention can be used to determine the ESBL phenotype in bacteria. The method of the present invention has an advantage over current phenotype tests in that the method suggested herein can be used not only with bacterial suspensions but also with bacterial lysates. The use of bacterial lysates can counteract potential bias due to resistance based on other mechanisms, in particular, decreased entry and increased efflux of the drug. Bacterial lysates cannot be used with current phenotype ESBL confirmatory tests because these tests rely on the growth of bacteria.

[0105] The method of the present invention can also be used as a high-throughput screening method for new β-lactamase inhibitors for the pharmaceutical industry. Currently, the β-lactamase inhibitors clavulanic acid and tazobactam are used in combination with aminopenicillins and piperacillin, respectively, to overcome the problem posed by bacteria producing β-lactamases. The clavulanic acid or tazobactam inhibits the activity of the β-lactamase, while the aminopenicillin or piperacillin kills the bacteria. Given the growing problem of ESBLs, there is a need in the pharmaceutical industry for a tool to screen for new compounds that inhibit ESBLs. The present invention is highly suitable for this purpose.

[0106] The present invention relates to the following embodiments:

[0107] 1. A method for characterizing antibiotic resistance of a microorganism, the method comprising the steps of:

[0108] a) providing a reference mass spectrum of an antimicrobial compound, of a modified product thereof, of a molecular target thereof, or of a modified enzyme substrate compound thereof;

[0109] b) exposing a microorganism, a cell lysate thereof, or a growth medium supernatant thereof to the antimicrobial compound or the substrate compound in an aqueous liquid, thereby providing an exposed sample;

[0110] c) obtaining a mass spectrum of the exposed sample;

[0111] d) comparing the mass spectrum obtained in step c) with the reference mass spectrum in step a), and

[0112] e) determining from the comparison whether a modification of the antimicrobial compound, of the modified product thereof, of the molecular target thereof, or of the substrate occurs after the exposure, and establishing that the microorganism potentially has resistance to the antimicrobial compound when the modification is observed.

[0113] 2. The method according to embodiment 1, wherein the modification comprises an enzymatic inactivation or degradation of the antimicrobial compound or the substrate and / or a methylation or overproduction of a molecular target thereof.

[0114] 3. The method according to embodiment 2, wherein the enzymatic degradation is caused by a β-lactamase.

[0115] 4. The method according to embodiment 3, wherein the β-lactamase is selected from the group consisting of β-lactamases of class A and class D according to the Ambler classification or β-lactamases belonging to class 2 according to the Bush classification.

[0116] 5. The method according to embodiment 4, wherein the β-lactamase is an extended spectrum β-lactamase (ESBL).

[0117] 6. The method according to any of the preceding embodiments, wherein the microorganism is a microorganism suspected to produce an ESBL, preferably selected from the group consisting of Klebsiella pneumoniae, Escherichia coli, Klebsiella oxytoca and Proteus mirabilis.

[0118] 7. The method according to any of the preceding embodiments, wherein the antimicrobial compound is a β-lactam antibiotic, preferably selected from the group consisting of penicillins, cephalosporins, cephamycins and carbapenems, more preferably selected from the group consisting of ceftazidime, cefotaxime, ceftriaxone, cefpodoxime and aztreonam.

[0119] 8. The method according to any of the preceding embodiments, wherein the method is also part of a method for characterizing an antimicrobial modifying enzyme of a microorganism, preferably the antimicrobial modifying enzyme is an extended spectrum β-lactamase (ESBL).

[0120] 9. The method according to embodiment 8, wherein the method for characterizing the enzyme comprises determining the rate of modification, preferably degradation, of the antimicrobial compound or the substrate compound and / or the rate of production of the enzymatic modification product of the compound or substrate or the rate of production of the molecular target of the compound, thereby determining the Michaelis-Menten (Km) constant and the maximum reaction rate (Vmax) of the enzyme.

[0121] 10. The method according to any of the preceding embodiments, wherein after step b) the exposed sample is coated together with the matrix on a mass spectrometry sample carrier, wherein the sample is dried on the sample carrier to prepare a mass spectrometry sample for matrix assisted laser desorption ionization mass spectrometry analysis (MALDI-MS).

[0122] 11. The method according to embodiment 10, wherein the mass spectrometry is obtained by using MALDI triple quadrupole MS.

[0123] 12. The method according to any of the preceding embodiments, wherein the exposed sample is a crude cell lysate of the microorganism exposed to the antimicrobial compound.

[0124] 13. The method according to any of the preceding embodiments, wherein in step (b) the microorganism is quantitatively analyzed by quantitatively analyzing in the sample one or more structural biomolecules or metabolites derived from the microorganism, preferably wherein the structural biomolecule or metabolite is a DNA molecule.

[0125] 14. A kit for characterizing antibiotic resistance of a microorganism, comprising:

[0126] a) a lysis buffer for lysing a microorganism;

[0127] b) at least one antimicrobial compound or a substrate of an antimicrobial compound modifying enzyme, and

[0128] c) a MALDI matrix,

[0129] preferably the kit further comprises:

[0130] d) a carrier carrying the at least one antimicrobial compound or substrate, wherein the carrier is optionally a disposable mass spectrometry sample carrier.

[0131] 15. A system for characterizing antibiotic resistance of a microorganism, comprising:

[0132] at least one antimicrobial compound or a substrate of an antimicrobial compound modifying enzyme;

[0133] a container for exposing a microorganism, a cell lysate thereof, or a supernatant of a growth medium thereof to the at least one antimicrobial compound in an aqueous solution, preferably wherein at least one substrate compound is provided in the container;

[0134] a lysis buffer for lysing the microorganism;

[0135] a MALDI matrix;

[0136] - a mass spectrometry analysis device;

[0137] - a reference mass spectrum of an antimicrobial compound, of an enzymatic modification product thereof, of a molecular target thereof or of a modified enzyme substrate compound, and

[0138] - a mass spectrometry analysis sample carrier,

[0139] optionally further comprising

[0140] - an automated pipettor for liquid handling;

[0141] - a computer program comprising computer program code means which, when said program is run on a computer, or wherein said computer program is embodied on a computer readable medium, is able to perform all the steps of any one of embodiments 1-13.

[0142] 16. A computer program comprising computer program code means which, when said program is run on a computer, is able to perform all the steps of any one of embodiments 1-13.

[0143] 17. A computer program product comprising computer program code means stored on a computer readable medium which, when said program product is run on a computer, is able to perform the method of any one of embodiments 1-13. EMBODIMENTS

[0144] We detected the activity of β-lactamases in crude lysates of E. coli producing CTX-M-1 and CTX-M-9 and K. pneumoniae producing SHV-2 by using benzylpenicillin as substrate. With benzylpenicillin as substrate we were able to monitor the enzyme kinetics of pure penicillinase (derived from B. Cereus).

Claims

1. A method for determining whether microorganisms in a sample are modified with antimicrobial compounds, their molecular targets, or substrate compounds of their modified enzymes, the method comprising the following steps: a) Using a computer program, the mass spectra of a sample obtained by exposing the microorganism, its cell lysate, or its growth medium supernatant to an aqueous solution of the antimicrobial compound or the substrate compound are compared with the reference mass spectra of the antimicrobial compound, its enzyme-modified product, its molecular target, or the substrate compound of the modified enzyme. and b) Using a computer program, determine from the comparison whether modification of the antimicrobial compound or its molecular target or substrate compound occurs after the exposure.

2. The method of claim 1, wherein the modification comprises enzymatic inactivation or enzymatic degradation of the antimicrobial compound or the substrate and / or methylation or overproduction of its molecular target.

3. The method of claim 2, wherein the enzymatic degradation is caused by β-lactamase degradation.

4. The method of claim 3, wherein the β-lactamase is selected from β-lactamases of classes A and D according to the Ambler classification, or β-lactamases belonging to class 2 according to the Bush classification.

5. The method of claim 4, wherein the β-lactamase is an extended-spectrum β-lactamase (ESBL).

6. The method according to any one of the preceding claims, wherein the microorganism is a microorganism suspected of generating ESBL.

7. The method according to any one of the preceding claims, wherein the antimicrobial compound is a β-lactam antibiotic.

8. The method of claim 7, wherein the antimicrobial compound is selected from penicillins, cephalosporins, cephamycins and carbapenems, more preferably from ceftazidime, cefotaxime, ceftriaxone, cefpodoxime and aztreonam.

9. The method according to any one of the preceding claims, wherein the method is also part of a method for characterizing antimicrobial modifying enzymes of microorganisms, preferably the antimicrobial modifying enzyme being an extended-spectrum β-lactamase (ESBL).

10. The method of claim 9, wherein the method for characterizing the enzyme comprises determining the modification rate of the antimicrobial compound or the substrate compound and / or the generation rate of the enzyme-modified product of the compound or the substrate, or the generation rate of the molecular target of the compound, thereby determining the Mie-Mann (Km) constant and the maximum reaction rate (Vmax) of the enzyme.

11. The method of claim 9, wherein the method for characterizing the enzyme comprises determining the degradation rate of the antimicrobial compound or the substrate compound and / or the generation rate of the enzyme-modified product of the compound or the substrate, or the generation rate of the molecular target of the compound, thereby determining the Mie-Mann (Km) constant and the maximum reaction rate (Vmax) of the enzyme.

12. The method of claim 2, wherein the overproduction of the molecular target is detected by using an internal standard and observing an increase in the ratio of the molecular target to the internal standard.

13. The method of claim 12, wherein the internal standard is a nucleic acid.

14. The method of claim 6, wherein the microorganism is selected from Klebsiella pneumoniae, Escherichia coli, Klebsiella oxytoca, and Proteus mirabilis.

15. The method of claim 1, wherein the mass spectrum of the sample is obtained by coating the exposed sample and the matrix together on a mass spectrometry sample carrier plate and drying the sample on the sample carrier plate to generate a mass spectrometry sample for matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS).

16. The method of claim 15, wherein the mass spectrometry is obtained by using MALDI triple quadrupole MS.

17. The method of claim 1, wherein the exposed sample is a crude cell lysate of the microorganism exposed to an antimicrobial compound.

18. The method of claim 1, wherein the microorganism is quantitatively analyzed by quantitative analysis of one or more structural biomolecules or metabolites derived from the microorganism in the sample.

19. The method of claim 18, wherein the structural biomolecule or metabolite is a DNA molecule.

20. The method of claim 1, wherein the mass spectrometry is obtained by using one of MALDI triple quadrupole mass spectrometry, MALDI-TOF mass spectrometry, and MALDI-FT-ICR mass spectrometry.

21. The method of claim 1, wherein the exposed sample is a body fluid or tissue sample of a human or animal subject suspected of carrying microorganisms.

22. The method of claim 1, wherein the antimicrobial compound is one of erythromycin, ciprofloxacin, vancomycin, methicillin, and tetracycline.

23. The method of claim 1, wherein the modification of the molecular target is RNA methylation.

24. A system comprising: - At least one antimicrobial compound or an enzyme substrate modified by an antimicrobial compound; - A container for exposing microorganisms, their cell lysates, or the supernatant of their growth medium to an aqueous solution of at least one antimicrobial compound; - A lysis buffer solution for lysing the microorganisms; -MALDI matrix; -Mass spectrometry analysis device; - Reference mass spectra of antimicrobial compounds, their enzyme-modified products, their molecular targets, or their modified enzyme substrates, and -Mass spectrometry analysis sample carrier plate - A computer program comprising computer program code tools, which, when run on a computer or when the computer program is implemented on a computer-readable medium, can perform all the steps of any one of claims 1-23; Optionally further include - An automated pipette used for liquid handling.

25. The system of claim 24, wherein the substrate of the at least one antimicrobial compound or the antimicrobial compound-modified enzyme is provided in the container.

26. The system of claim 24, wherein the at least one antimicrobial compound is at least one β-lactam antibiotic, preferably selected from penicillins, cephalosporins, cephamycins and carbapenems.

27. The system of claim 26, wherein the at least one antimicrobial compound is selected from ceftazidime, cefotaxime, ceftriaxone, cefpodoxime, and aztreonam.

28. The system of claim 26, further comprising a β-lactamase inhibitor.

29. The system of claim 28, wherein the β-lactamase antibiotic is aminopenicillin or piperacillin, and the β-lactamase inhibitor is clavulanic acid or tazobactam accordingly.

30. The system of claim 24, wherein the MALDI mass spectrometry device is a MALDI triple quadrupole mass spectrometer, a MALDI-TOF mass spectrometer, and a MALDI-FT-ICR mass spectrometer.

31. A computer program product comprising a computer-readable medium storing computer program code tools for performing the method of any one of claims 1-23.

32. An exposed sample prepared by exposing microorganisms, their cell lysates, or the supernatant of their growth medium to an aqueous solution on a mass spectrometry sample carrier plate using an antimicrobial compound.

33. A reagent kit comprising: a) Lysis buffer for lysing microorganisms; b) At least one antimicrobial compound or a substrate of an enzyme modified by an antimicrobial compound, and c) MALDI matrix Preferably, the kit further comprises: d) A carrier carrying at least one antimicrobial compound or substrate, wherein the carrier is optionally in the form of a sample plate for primary mass spectrometry analysis.