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    • 专利摘要:
    MEASUREMENT WITH MASS SPECTROMETRY OF BETA-LACTAMASE RESISTANCE. The invention relates to the determination of the resistance of microorganisms that produce (Beta) -lactamases, in particular "extended-spectrum beta-lactamases" (ESBL). The invention provides a method by which microbial resistance can be measured in a very simple and fast way through the catalytic effect of (Beta) -lactamases produced by microbes on (Beta) - lactam antobiotics, which consists of a hydrolytic cleavage of (Beta) -lactamic ring. The method determines the resistance of the bacteria a few hours after a suitable substrate, an anti-biotic (Beta) -lactam or an adapted (Beta) -lactam derivative, has been added to a suspension of the microbes, by direct measurement by spectrometry of mass of the substrate breakage caused by (Beta) -lactamases.
    公开号:BR112012031558B1
    申请号:R112012031558-7
    申请日:2011-06-10
    公开日:2020-12-08
    发明作者:Markus Kostrzewa；Karsten Michelmann；Katrin Sparbier
    申请人:Bruker Daltonik Gmbh；
    IPC主号:
    专利说明:

    Field of Invention
    [0001] The invention relates to the determination of the resistance of β-lactamase-producing bacteria, including "extended-spectrum β-lactamasees" (ESBL). Prior art
    [0002] Many types of microbes, mainly single-celled bacteria and fungi, can be identified quickly and easily by mass spectrometry by transferring small amounts of microbes from a colony grown in the usual way or in a nutrient medium, to a mass spectrometry sample support plate, in which they are prepared with a solution of a matrix substance and measured with mass spectrometry after ionization by matrix-assisted laser desorption. The mass spectrum shows the masses and intensities of the characteristic proteins, if they are present in the microbes in sufficient concentration. This spectrum, which always displays peaks of approximately 40 to 80 microbial proteins at a time, is used to determine its identity by similarity analyzes with thousands of reference spectra in corresponding spectral libraries. Here, the term "identification" denotes a taxonomic classification, that is, the determination of the family, genus and species. The research is being carried out in many locations to bring together trusted and legally approved libraries for medical use (so-called "validated" libraries) with reference spectra of thousands of microbes.
    [0003] This method of identifying microbes has proved extraordinarily successful both in studies and in the daily routine of many microbiological laboratories. It is fast, low cost and has very low error rates, much lower than conventional methods of microbiological identification.
    [0004] Special versions of these methods can be used to identify not only the species of the microbe, but, not infrequently, also the subspecies and, in some cases, even the specific cepases, if they are different from the most frequent in terms of masses or intensities and, therefore, proteins detectable by mass spectrometry. For a more detailed description, see patent application DE 10 2009 032 649 A1 (T. Maier and M. Kostrzewa, 2009, US 2011/0012016 A1; GB 2 471 746 A), for example, which presents not only a detailed explanation of the method, but also a more refined identity search.
    [0005] The identification of microbes plays a special role in dealing with infectious diseases, particularly sepsis. In this case, in order to apply the correct medical treatment immediately, it is important to be able to identify the pathogen species very quickly. Mass spectrometry identification has been tried and tested in these cases as well, and is on track to gain acceptance in clinical and microbiological laboratories.
    [0006] In the field of medicine, however, there is not only the problem of rapid identification, but also the problem of detecting resistance to antibiotics commonly used. Without knowing the resistance, rapid control of the disease is not possible. Therefore, it is necessary not only to make a quick identification, but also to determine and characterize the resistance of microorganisms promptly. Some species of microbes are known to be almost completely resistant to certain antibiotics, so there is no reason to determine resistance after accurate identification. In most cases, however, one species has strains that are not resistant, others with little resistance and there are, in particular, still others, highly resistant and with different resistance to different types of antibiotics. For this reason, it is essential to determine the type and power of resistance.
    [0007] It seems obvious to use mass spectrometers, not only for taxonomic identification, but, moreover, to determine the resistance of microbes, especially bacteria, to certain antibiotics. However, this task has proven to be very difficult. Although the resistances must be expressed by the presence of new or modified proteins, until now there is no evidence that it is possible to identify them directly in the protein profile measured by mass spectrometry. After all, out of hundreds or even thousands of microbes' proteins, only 40 to 80 are measured in the mass spectrum. Resistance, therefore, must be determined indirectly. A first attempt to determine resistance is presented in DE 10 2006 021 493 B4 (V. Govorun and J. Franzen; GB 2 438 066 B; US 2008/0009029 A1); however, this method has so far not been accepted. The method is essentially based on a change in protein profiles, caused by cell death after the addition of antibiotics, or on the determination of a growth cessation compared to the resistant reference microbes.
    [0008] Antibiotic resistance identifies characteristics of microorganisms (here, predominantly bacteria) that allow them to weaken or completely neutralize the effect of substances with antibiotic activity. Resistances are now widespread; in the USA approximately 70% of hospital-acquired infectious germs are resistant to at least one antibiotic. Patients are often infected with strains of bacteria resistant to various antibiotics (multiple resistance). The supergerms are methicillin-resistant Staphylococcus aureus, known by the acronym MRSA, species of the genus Pseudomonas, ESBL-resistant Escherichia coli and Mycobacterium tuberculosis. Assessments carried out by the Center for Disease Control and Prevention CDC (Center for Disease Control and Prevention) consider that two million infections were acquired in hospitals in the USA in 2004, resulting in approximately 90,000 deaths, much more than the number of deaths caused by traffic accidents, domestic or industrial.
    [0009] In daily use, antibiotics, medicines or pharmaceuticals are generally understood to treat bacterial infectious diseases. The great success of antibiotics in medicine began with penicillin. The success of penicillin, and also the appearance of the first resistances, led researchers to look for and verify many other antibiotics: streptomycin, chloramphenicol, aureomycin, tetracycline and many others. Most of the antibiotics known today are derived from natural substances. Colloquially, penicillin is nowadays synonymous with antibiotics.
    [00010] Penicillin is an e-lactam antibiotic. Β-lactams act by binding to penicillin-carrying proteins (PBP), a peptidoglycan transpeptidase responsible for the formation of peptide chains that strengthen cell walls. The chains between β-lactams and PBP make PBP ineffective. The lack of sufficient amounts of effective PBP causes damage to the cell wall when bacteria are growing; thus, the membrane loses control of its permeability and can no longer regulate the cytoplasm concentration. After a short time, the bacteria becomes unable to live. Under extreme conditions, it is possible to observe bacteria cells literally "burst" in the laboratory. This is how β-lactams act as bactericides.
    [00011] Since the first applications of penicillin, bacteria have developed increasingly different types of resistance. An important type of bacterial resistance to e-lactams is the formation of enzymes (β-lactamases), which catalytically open the e-lactam ring by hydrolysis, and thus render it ineffective. Currently, more than 340 variants of β-lactamases are known, formed by many types of bacteria. They can be divided into different categories according to their general structure or how they act. The genetic information for the synthesis of the enzyme, which is initially produced by mutations, is transmitted by chromosomes or plasmids. Plasmid information can be transferred between bacteria by various mechanisms, even by contact between bacteria of different species ("horizontal transfer"). Depending on the action of β-lactamases, a distinction is made between penicillinases and cephalosporinases, but there are still other categories. The catalytic effect of these enzymes makes a small amount of β-lactamases sufficient to destroy large amounts of e-lactam antibiotics.
    [00012] Today there are a large number of β-lactam antibiotic derivatives, including several penicillins (benzylpenicillins, oral penicillins, aminopenicillins, isoxazolylpenicillins, ureidopenicillins), cephalosporins, monobactams and carbapenemas. These are generally converted into a derivative with larger chemical groups in order to sterically prevent β-lactamases. Extended-spectrum β-lactamases (ESBL), in turn, can cleave a wide variety of e-lactam antibiotics. ESBLs were initially formed by β-lactamase mutation points. The genes for ESBL are in plasmids, which can be transferred horizontally from bacteria to bacteria.
    [00013] ESBL-producing bacteria are resistant to penicillins, cephalosporins (generations 1-4) and monobactams. Escherichia coli and Klebsiella (Gram-negative bacteria) are the most common bacteria producing ESBL genes, but microbiologists are watching the rapid spread of the ESBL resistance mechanism with great concern. In addition to the resistance of Staphylococcus aureus to methicillin (MRSA), ESBL is one of the concerns that most plague research on infections.
    [00014] β-lactamase inhibitors are a tool against β-lactamases, being administered together with e-lactams to weaken the effect of β-lactamases that are present in bacteria. Consecrated combinations are clavulanic acid + amoxicillin, sulbactam + ampicillin, tazobactam + piperacillin. Not all combinations have the ideal effect. These tools should be used only after careful identification of the bacteria and careful determination of their resistance, since it can be expected that they too quickly become inept.
    [00015] In the 1970s and 1980s there was still a lot of research activity in the field of antibiotics. Although antibiotics are among the most commonly prescribed drugs in the world, at the present time the development of new antibiotics has slowed considerably; with a 13% market share, they form the largest specific segment of pharmaceutical products we use. Of the approximately 8,000 antibiotic substances known today, only around 80 are used therapeutically, especially because of side effects, but also because of approval costs. In Germany, in 2005, according to the German Federal Institute for Medicines and Medical Devices (BfArM), a total of 2,775 antibiotics were approved, but that number covers only about 80 antibiotic substances.
    [00016] Over time, dealing with the problem of microorganisms resistant to antibiotics such as bactericides or fungicides, is becoming increasingly imperative. On the one hand, there is the speed with which microorganisms form resistance to different types of antibiotics; on the other hand, fewer and fewer new antibiotics for medical applications are being developed. Since many new antibiotics are due to be withdrawn from the market after a short time due to inefficiency, it is increasingly difficult and less profitable for pharmaceutical companies to invest significantly in the development of new antibiotics. According to the WHO, between 1990 and 2005 only three new active antibiotic substances were launched, compared with ten between 1940 and 1950 and five between 1971 and 1980.
    [00017] The reasons for the rapid development of resistance are multiple: irresponsible prescription of antibiotics, even when there is no need; treatments with bactericides irresponsibly interrupted before the infectious agent is completely wiped out; irresponsible, and often purely preventive, consumption in agriculture and livestock. All of these practices influence the selection and propagation of resistant microbial species compared to non-resistant species.
    [00018] Celebrated in the middle of the last century as the great hope in the fight against infectious diseases, antibiotics are quickly becoming an ineffective tool. The only hope of avoiding this is through administrations directed at treatments that must be fully completed, which requires the rapid identification of infectious pathogens, as well as the rapid identification of their specific resistance to different types of antibiotics. Purpose of the invention
    [00019] The objective of the invention is to provide a method by which a resistance to different types of e-lactam antibiotics, associated with the production of β-lactamase by microbes, bacteria in particular, can be measured by mass spectrometry of simply and quickly. Summary of the Invention
    [00020] The invention provides a method by which a microbial resistance as a function of β-lactamases can be measured very easily and quickly with a mass spectrometer. The method determines the resistance of the bacteria a few hours after the microbes are associated with an appropriate substrate, an β-lactam antibiotic or an adapted e-lactam derivative, by direct measurement by mass spectrometry of the hydrolytic attack of β-lactamases on the substrate. . The catalytic effect of β-lactamases produced by bacteria on the substrate causes the e-lactam ring to rupture by hydrolysis. The amount of substrate is decreased and the product of the hydrolytic cleavage appears in place, its mass being heavier by 18 units of atomic mass.
    [00021] The reaction of enzymatic cleavage is quite rapid; as long as it is not prevented by the gradual lack of substrate, it lasts approximately between one and one hundred milliseconds per molecular reaction, with characteristic differences for different β-lactamases. The measurement of the reaction rate provides initial information about the type and power of β-lactamases.
    [00022] In principle, the measurement can be performed with any mass spectrometer, however it is especially favorable to use the same MALDI time-based mass spectrometer that was used for the identification of bacteria. Since the MALDI process (matrix-assisted laser ionization and desorption) for the ionization of substances in the lower mass range of a few hundred units of atomic mass generates a very pronounced chemical background, it is favorable to use substrates that are found in regions with low bottom. This can be accomplished by producing adapted substrates with molecular weights between 700 and 1200 atomic mass units. It is also advantageous to increase the protonic affinity of the substrates, in order to increase their degree of ionization. However, it is often advantageous to be able to use higher concentrations of the substrate to increase sensitivity. So that a high bactericidal effect of high concentration does not kill bacteria immediately, the substrates can be adapted so that their antibiotic effect, that is, their MIC value, is relatively small. MIC is the "minimum inhibitory concentration" for inhibition by β-lactamase inhibitors in the presence of the corresponding antibiotic, and serves as a measure of the strength of the resistance and the strength of the antibiotic.
    [00023] An advantageous embodiment of a substrate receives by covalent bonding a tag of six histidines (6 * His-tag) to a β-lactam, for example. A 6 * His-tag consisting of a chain of six histidine molecules, increases the molecular mass by approximately 800 atomic mass units, improves the proton affinity and makes it possible to extract the substrate and its cleavage product in pure form liquid reaction. This extraction can, for example, be carried out with the help of commercially available magnetic spheres, in which there is a chelate charged with nickel ions that reversibly binds to a 6 * His-tag. The preparation of the MAL-DI sample can then be carried out, whose matrix substance contains the remaining substrate and its cleavage product in a highly enriched and purified form, thus allowing a very sensitive measurement.
    [00024] It is possible, in particular, to develop specific tests of varying strengths, with the introduction of different types of substrates, with the different substrates being adapted in such a way and introduced in such concentrations, that its breaking pattern allows the identification of the category and also the strength of the action of β-lactamases. The substrates can mimic accessibility to the β-lactam ring of different groups of antibiotics, for example. Brief description of the Figures
    [00025] The upper mass spectrum in Figure 1 shows the effect of mixing ampicillin, with a molar mass of 349.41 atomic mass units, to a suspension of E. coli of the DH5alpha strain that has no resistance. There is no breakdown, either of ampicillin (in this case, mass of 350 atomic mass units), or of ampicillin sodium salt (mass of 372 atomic mass units). The lower mass spectrum, on the other hand, exhibits the effect of an E. coli strain with ESBL resistance mechanism to ampicillin and sodium salt: both are broken down, resulting in mass hydrolysis products 368 and 390 mass units atomic. Preferred configuration
    [00026] The invention provides a very simple and fast method of determining microbial resistance based on the production of β-lactamases by microbes, especially by bacteria.
    [00027] The method basically adds one or more suitable substrates to a suspension of bacteria. The substrates can be e-lactam antibiotics or e-lactam derivatives, preferably adapted. If the bacteria have a resistance to β-lactamase, at least one substrate is broken by β-lactamase under suitable conditions of incubation within minutes to hours, with hydrolysis opening of the e-lactam ring. This hydrolytic breakdown of the substrate by β-lactamases can be directly measured by mass spectrometry. The amount of substrate decreases and is replaced by the product of hydrolyzed cleavage, the mass of which is 18 units of heavier atomic mass.
    [00028] In Figure 1, this break, which occurs only when there is resistance, is shown using the e-lactam antibiotic ampicillin in two mass spectra. The upper mass spectrum shows the result of mixing ampicillin, with a molar mass of 349.41 atomic mass units, to a suspension of E. coli from the strain DH5alfa. This strain has no resistance at all. Therefore, no breakdown, either of ampicillin (visible here in the mass of 350 atomic mass units), or of the sodium ampicillin salt (with mass of 372 atomic mass units) is observed. The lower mass spectrum, on the other hand, exhibits the effect of an E. coli strain with ESBL resistance mechanism to ampicillin and sodium salt: both are broken down, resulting in mass hydrolysis products 368 and 390 mass units atomic.
    [00029] Ampicillin is a semi-synthetic pharmaceutical product with antibiotic activity belonging to the group of β-lactam antibiotics (penicillins). It is known as a broad spectrum antibiotic due to its effectiveness against Gram-positive pathogens and some Gram-negative rods. In chemical terms, ampicillin is an aminopenicillin.
    [00030] As with all e-lactam antibiotics, the bactericidal effect (which destroys bacteria) of ampicillin is based on the blocking of an enzyme, D-alanine transpeptidase, which is present in different bacteria in different forms. This enzyme is necessary for the formation of a new and firm cell wall in the phase of division or growth of bacteria. These transpeptidases are also called penicillin-binding proteins (PBP). Blocking occurs by coupling, with the e-lactam ring acting on the receiving structure because it has the coupling pattern. Coupling prevents new synthesis of rigid cell walls. The cells are therefore unable to divide, but continue to live for a time until their growth results in a sufficiently high number of cell wall lesions to cause the cell to die. However, the division and growth of human cells are not prevented, as human cells have only one cell membrane, but no cell wall and, therefore, no corresponding transpeptidase.
    [00031] In the example shown in Figure 1, 10 microliters of ampicillin solution with a concentration of 10 milligrams per milliliter of water was added to an Eppendorf test tube. Three colonies of the bacterium to be tested were chosen, and were put back in suspension in the 10 microliters of ampicillin solution. The vessels were incubated for three hours at 37 ° C with shaking. After incubation, they were centrifuged for two minutes at 13,000 revolutions per minute, to separate the cells. The remaining ampicillin and the hydrolyzed reaction product are now in the supernatant.
    [00032] In principle, the measurement can be performed with any mass spectrometer, but it is especially favorable to be able to use the same MALDI time-based mass spectrometer that was used for the identification of bacteria. For this purpose, 1.5 microliters of the supernatant was applied to the mass spectrophotometric support. After drying, the samples were covered with a microliter of a matrix solution. The matrix used was α-cyano-4-hydroxycinaminic acid (HCCA) at a concentration of 10 milligrams per milliliter in a mixture of water, 50% methyl acetonitrile cyanide and 2.5% trifluoroacetic acid. After drying, a mass spectrum is acquired from this preparation on the mass spectrometer by MALDI flight time in the usual way.
    [00033] The concentration of ampicillin used as a substrate for this example is extraordinarily high, more than a thousand times greater than would be necessary for a therapeutic treatment. The fact that this amount of ampicillin is broken, demonstrates the extraordinary effectiveness of extended-spectrum β-lactamases (ESBL). It can hardly be assumed that bacteria will survive this high concentration for a long time; however, the small amount of β-lactamase expelled during its lifetime is sufficient to catalytically break the large amount of substrate. The high concentration was chosen so that the signals can be seen clearly above the high chemical background that exists in this mass range. The concentration could be a factor 100 times lower if a substrate in the range of approximately 800 to 1000 atomic mass units could be used, made possible by adapted substrates with high molecular weight.
    [00034] It is also advantageous to increase the protonic affinity of the substrates, in order to increase the ionization yield. Β-lactams with their low masses do not have a high proton affinity; therefore, only small proportions of them are ionized in the ionization process. The sensitivity can be increased by 10 times, inserting amino acids with high protonic affinity, for example.
    [00035] However, it is often advantageous to use higher concentrations of substrate to further increase sensitivity. However, to prevent these bacteria with less strong β-lactamases from being killed immediately by high bactericidal efficacy, the substrates can be adapted so that their antibiotic effect, that is, their MIC value, is relatively small. The effectiveness of the antibiotic usually decreases as the size of the molecules increases, as they are significantly prevented from penetrating through the pores in the cell wall in bacteria.
    [00036] In addition, it is advantageous to adapt the substrates so that they can be completely and easily extracted from the supernatant. To this end, anchor groups can be provided in which immobilized partners can be used to extract them. The coupling of a biotin group to the substrate is described here as a first example. The substrate and the degradation product can be extracted from the supernatant by streptavidin which is immobilized on the walls. Since the connection between biotin and streptavidin is reversible, the substrate and its degradation product can be processed and measured after being enriched in the known way. Suitable receptacles whose interior walls are coated with streptavidin are commercially available, as are coated microparticles, such as magnetic beads.
    [00037] A particularly advantageous embodiment of an extractable substrate is given by the covalent attachment of a 6 * His-tag to a β-lactam, for example. A 6 * His-tag is composed of six molecules of histidine, which increases the molecular weight by approximately 800 atomic mass units, improves the proton affinity and provides an easy process for the extraction of the substrate and its product from cleavage of the reaction liquid . This extraction can be carried out, for example, with magnetic spheres. Magnetic spheres coated with chelates are commercially available. These chelates can be charged with nickel ions. Nickel ions bind reversibly to 6 * His-tags. This makes it easy to perform a MALDI sample preparation in the known way, with samples containing only the remaining substrate and its cleavage product in a purified form, fixed in crystals of the matrix substance, which allows for a very sensitive measurement.
    [00038] The enzymatic reaction of cleavage of β-lactamases is quite fast; as long as it is not impeded by the gradual lack of substrate, it takes approximately one to one hundred milliseconds per molecular reaction. The characteristic differences in the reaction rates of different β-lactamase can be measured, and provide information about the power of β-lactamase and therefore also indicate the type of β-lactamase. In the most favorable case, the reaction speed can be measured in a single mass spectrum. If the incubation is stopped after exactly half an hour, for example, the relationship between the remaining substrate and the degradation product can be used to read the reaction speed if the method is calibrated accordingly.
    [00039] Another advantageous modality is to use several different substrates, adapted in a single test of varying strengths. The substrates can, for example, be supplied with different types of steric impediments to attack by β-lactamases, as they are present in different antibiotics. From the pattern of the break and its speed, conclusions can be drawn about the type of β-lactamases and the efficacy of different types of antibiotics. Using suitable substrates and choosing the right concentrations, it is possible to determine how effective β-lactamases are. A simple example for the simultaneous breaking of two substrates (ampicillin and its sodium salt) is shown in Figure 1, although no substrate prepared to measure with different resistance against breaking has been used in this case.
    [00040] In particular, microbial resistance measurement can be used for microbes that can be obtained in pure form from blood or blood cultures, as explained in DE 10 2009 033 368 A1 (T. Maier; WO 2011 / 006911 A3), for example.
    [00041] Instead of a matrix assisted laser ionization and desorption (MALDI) in a MALDI time-of-flight mass spectrometer, it is obviously possible to use other types of ionization, such as electrospray ionization (ESI) and other types of spectrome - mass spectrums, such as the orthogonal ion injection time-of-flight mass spectrometer (OTOF), cyclotronic ion resonance mass spectrometers (ICR-MS), Kingdon electrostatic mass spectrometers, or in particular, low cost ion trap mass spectrometers, to analyze substrate breakage. Experts in the field are familiar with all of these mass spectrometers and ionization methods, so we will avoid detailed explanations here.
    [00042] A particularly suitable option for measuring substrate breakdown and increase in breakdown product is a quadruple triple mass spectrometer, which essentially makes only a comparative measure of substrate and breakdown product. This triple quadruple mass spectrometer can achieve extremely high sensitivity, so that very small amounts of substrate are sufficient for this method.
    [00043] In order to simplify the determination of resistance, some or all of the necessary materials can be supplied in sterile consumable packages (kits). In particular, consumable packages can contain exact quantities of adapted substrates and, if necessary, also the corresponding matrix substances. In addition, they may contain disposable MALDI mass specimen sample holders. Consumable product packages can be produced commercially.
    [00044] Mass spectra can be evaluated visually and also by means of suitable computer programs. It is possible to develop and use programs for the evaluation of tests of varied resistance. These programs can immediately determine the type and strength of the microbes' β-lactamase resistance from the break pattern, and present the suggested treatments.
    权利要求:
    Claims (14)
    [0001]
    1. Method for determining a microbial e-lactam resistance, based on the production of β-lactamases by the microbes, characterized by the fact that the microbes are placed next to a substrate and the enzymatic breakdown of the substrate by the β-lactamases of the microbes is measured by mass spectrometry through the acquisition of a mass spectrum of the remaining substrate and the degradation product.
    [0002]
    2. Method according to claim 1, characterized by the fact that the substrate molecules contain a β-lactam ring.
    [0003]
    3. Method according to claim 2, characterized by the fact that the substrate is an e-lactam antibiotic or an e-lactam derivative.
    [0004]
    4. Method, according to claim 1, characterized by the fact that the substrate has a molecular weight between 700 and 1200 atomic mass units.
    [0005]
    5. Method according to claim 1, characterized by the fact that the substrate has only a weak antibiotic effect.
    [0006]
    6. Method, according to claim 1, characterized by the fact that the substrate molecules have an anchor group, which can be used to extract them from the solutions.
    [0007]
    7. Method according to claim 6, characterized by the fact that the anchor group is the biotin group or a 6 * His-tag.
    [0008]
    8. Method, according to claim 1, characterized by the fact that the breakage of the various types of substrate is measured simultaneously.
    [0009]
    9. Method, according to claim 8, characterized by the fact that different types of substrate are adapted so that their breaking pattern allows to identify the different categories of β-lactamases.
    [0010]
    10. Method according to claim 9, characterized by the fact that different substrates around the e-lactam ring mimic the steric forms of different antibiotics.
    [0011]
    11. Method, according to claim 1, characterized by the fact that the reaction rates of substrate breakage are measured.
    [0012]
    12. Method according to claim 1, characterized by the fact that the microbes were obtained from blood or a blood culture.
    [0013]
    13. Method, according to claim 1, characterized by the fact that the remaining substrate quantities and their breakdown product are measured by ion-assisted mass spectrometry and matrix-assisted laser desorption.
    [0014]
    14. Method for determining microbial e-lactam resistance, based on the production of β-lactamases by microbes, characterized by the fact that it comprises the following steps: (a) inclusion of microbes in a solution of at least one substrate, which can be broken by β-lactamases, (b) incubation of the solution at a specific temperature for a specified time, (c) separation of the solution with the remaining substrate and its microbial breakdown product, and (d) acquisition of a mass spectrum of the solution.
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    法律状态:
    2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according art. 34 industrial property law|
    2019-07-16| B06T| Formal requirements before examination|
    2020-08-11| B09A| Decision: intention to grant|
    2020-12-08| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 10/06/2011, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    DE102010023452A|DE102010023452B4|2010-06-11|2010-06-11|Mass spectrometric measurement of β-lactamase resistance|
    DE102010023452.4|2010-06-11|
    PCT/EP2011/059670|WO2011154517A1|2010-06-11|2011-06-10|MASS SPECTROMETRIC MEASUREMENT OF β-LACTAMASE RESISTANCES|
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