WO2012113699A1 - Détermination par spectrométrie de masse de l'effet inhibiteur de substances sur les bêta-lactamases - Google Patents

Détermination par spectrométrie de masse de l'effet inhibiteur de substances sur les bêta-lactamases Download PDF

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WO2012113699A1
WO2012113699A1 PCT/EP2012/052662 EP2012052662W WO2012113699A1 WO 2012113699 A1 WO2012113699 A1 WO 2012113699A1 EP 2012052662 W EP2012052662 W EP 2012052662W WO 2012113699 A1 WO2012113699 A1 WO 2012113699A1
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beta
mass
combination drug
antibiotic
mass spectrum
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Markus Kostrzewa
Ulrich Weller
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Bruker Daltonik Gmbh
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/18—Testing for antimicrobial activity of a material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2560/00—Chemical aspects of mass spectrometric analysis of biological material

Definitions

  • the invention relates to a method to determine the inhibitory effect of test substances on beta-lactamases and particularly the inhibitory efficiency of the inhibitor of an antibiotic combination drug comprising a beta-lactam antibiotic and an inhibitor.
  • antibiotic usually means a pharmacologically active substance for the treatment of bacterial infectious diseases.
  • penicillin but also the appearance of the first resistances, led researchers to search for and discover many more antibiotics.
  • Penicillin is a beta-lactam antibiotic.
  • the beta-lactam antibiotics are a group of antibiotics which have a four-membered beta-lactam ring in their structural formula. They bond to the penicillin binding protein (PBP), a peptidoglycan transpeptidase which is present in bacteria in different variants. This enzyme is necessary for the formation and renewing of a rigid cell wall in the division or growth phase of bacteria. The bonding of a beta-lactam antibiotic to the PBP causes the PBP to become ineffective.
  • the beta-lactam ring of the antibiotic represents the attachment motif.
  • the attachment prevents the re-synthesis of the cell wall; the bacteria are therefore unable to divide, but live on initially until their growth leads to a sufficiently large number of cell wall lesions to cause the death of the cell.
  • the division and growth of human cells are not impeded, however, because human cells have only a cell membrane, but no cell wall and have therefore no corresponding transpeptidase.
  • bacterial strains have increasingly developed various types of resistance, i.e. they have acquired charac- teristics which allow them to weaken or completely neutralize the effect of antibiotic substances. Resistances are now widespread; in the USA, around 70% of the infectious pathogens acquired in hospitals are resistant to at least one antibiotic. Patients are often infected with bacterial strains which are resistant to several antibiotics (multi-resistance). Problematic pathogens are mainly the methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas spec, Escherichia coli with ESBL resistance and Mycobacterium tuberculosis.
  • MRSA methicillin-resistant Staphylococcus aureus
  • Pseudomonas spec Escherichia coli with ESBL resistance
  • Mycobacterium tuberculosis Mycobacterium tuberculosis.
  • Acute situations such as a sepsis or a secondary infection during an already existing primary illness (or primary infection) can be life- threatening.
  • Targeted administration requires the pathogen or pathogens to be identified as fast as possible and as correctly as possible, as well as the rapid detemiination of their resistances to the different antibiotics.
  • the resistance of bacteria is nowadays determined by culturing the bacteria in vitro on a nutrient medium (e.g. an agar plate) or in a nutrient medium (e.g. a culture broth), to which an antibiotic is added. Whether the bacteria grow under the influence of the antibiotic, i.e. are resistant to the antibiotic, is determined visually, either by eye or automated with optical devices. The usual procedure is to test cultures with graduated concentrations of the antibiotic in order to determine the minimum inhibitory concentration (MIC).
  • a nutrient medium e.g. an agar plate
  • a nutrient medium e.g. a culture broth
  • the minimum inhibitory concentration designates the lowest concentration of a sub- stance at which the multiplication of a microorganism can no longer be perceived visually. Usually, that concentration of an antibiotic is detennined which just still inhibits the growth of a colony. Antibiotics can also be characterized via the minimum bactericidal concentration (MBC) at which just about 99.9% of the pathogens are killed within a given period of time. While the MIC can be determined in principle for every antibiotic, the MBC only makes sense for those which can develop not only an inhibitory, but also a destructive (bactericidal) effect. These are, for example, aminoglycosides, gyrase inhibitors, and penicillins. According to the strength of the resistance determined, the pathogens detected are termed S (sensitive), I (intermediate) or R (resistant).
  • the routine method based on culturing allows a simple but time-consuming determi- nation of the resistance, because the duration of the analysis is dictated by the growth rate of the bacteria.
  • An additional advantage of the routine method consists in the fact that the effi- ciency or inefficiency of an antibiotic against the bacteria of a sample is measured directly (functional test).
  • identification here is deemed to mean the taxonomic classification, i.e. the determination of family, genus and species.
  • identification of bacteria by mass spectrometric methods is de- scribed in detail in a scientific review article by van Baar, for example (FEMS Microbiology Reviews, 24, 2000, 193-219: "Characterization of bacteria by matrix-assisted laser desorp- tion/ionization and electrospray mass spectrometry").
  • MALDI Matrix Assisted Laser Desorption/Ionization
  • the similarity analysis involves assigning each reference spectrum an indicator, which is a measure of the agreement between the corresponding reference spectrum and the mass spectrum of the sample (see for example Jarman et al., Analytical Chemistry, 72[6], 1217-1223, 2000: "An algorithm for automated bacterial identification using matrix-assisted laser desorption/ionization mass spectrometry").
  • This method of identifying microorganisms has proven to be extraordinarily successful both in studies as well as in the daily routine in many microbiological laboratories. It is fast, low-cost and has very low error rates, far lower than conventional microbiological identification methods.
  • beta-lactamases enzymes which enzymatically modify the beta-lactam antibiotics, often by a hydrolytic cleaving of the ⁇ -lactam ring, and thus render them ineffective.
  • the catalytic effect means that a small quantity of the beta-lactamases is sufficient to render large quantities of beta-lactam antibiotics ineffective.
  • the genetic information for the synthesis of the enzyme, which originated from corresponding mutations, is passed on chromosomally or plasmid- encoded.
  • the plasmidic information can be exchanged between bacteria by various mechanisms, even between bacteria of different species and genera ("horizontal exchange").
  • beta-lactamases More than 340 variants of beta-lactamases are currently known, and they are formed by a large number of bacterial species. They are grouped into four classes (A to D) according to their molecular structure, and into groups and subgroups depending on how they act.
  • a to D beta-lactam antibiotics
  • a subgroup of the beta-lactamases comprises the so-called extended spectrum beta-lactamases (ESBL or ESBL bacterial strains), which have originated from beta-lactamase encoding genes in the plasmids by point mutations and can cleave a broad spectrum of beta-lactam antibiotics.
  • ESBL bacterial strains are resistant to penicillins, cephalosporins, (generations 1 -4) and monobactams. It is mainly E. coli and leb- siellae (Gram-negative bacteria) which carry ESBL encoding genes. Microbiologists are watching the rapid spread of this ESBL resistance with great concern. It is one of the most worrying concerns in infection research apart from the methicillin resistance of Staphylococcus aureus (MRSA).
  • MRSA methicillin resistance of Staphylococcus aureus
  • beta-lactamase inhibitors are administered together with a beta-lactam antibiotic as a combination drug in order to inhibit or at least weaken the effect of the beta-lactamases.
  • Beta-lactamase inhibitors currently have a beta- lactam ring, but usually have no appreciable antibacterial efficiency.
  • combination drugs with clavulanic acid Figure 1A
  • sulbactam Figure IB
  • tazobactam Figure 1C
  • beta-lactamase inhibitors which are used in the following fixed combinations: amoxicillin with clavulanic acid, ampicillin with sulbactam and piperacillin with tazobactam. Not all combinations have the optimum effect.
  • These adjuvants should only be used after careful identification of the bacteria and determination of their resistance, because it is to be expected that resistances to combination drugs will also be formed very quickly.
  • the objective of the invention is to provide a method with which the inhibitory effect of substances on beta-lactamases is determined quickly and with certainty, particularly the inhibitoiy effect of the inhibitor in a combination drug, and thus to provide a method for determining the beta-lactamase-based resistance of a bacterial sample to such a combination drug.
  • the invention relates to a method for determining the inhibitory effect of a test substance on beta-lactamases, wherein the test substance is brought together with a beta-lactamase and a substrate which has a beta-lactam ring, and afterwards a mass spectrum is acquired and evaluated as to whether the substrate has been enzymatically modified by the beta-lactamase.
  • the substance under investigation (test substance) and the beta-lactamase (or beta- lactamase producing microbes) are brought together with the substrate, preferably in a liquid reaction mixture. After a certain reaction time, the mass spectrum is acquired from the liquid or a portion of it.
  • the concentration of a substrate decreases and that of reaction products increases, so the mass signal of the substrate in the mass spectrum is small or completely absent, and one or more mass signals from reaction products of the substrate are present in the mass spectrum.
  • the decrease in the substrate as a result of an enzymatic modification is preferably derived from the intensity ratio of the mass signal of the substrate to the mass signal of a reaction product or to the mass signal of a reference substance.
  • the enzymatic activity of the beta-lactamase can also be derived from the mass signal of the substrate alone.
  • an additional mass spectrum is acquired from the mixture at the start time, in order to derive enzymatic modifications from the changed signal intensities of the corresponding mass signals in the two mass spectra.
  • the beta-lactamases used in the method can be artificially synthesized or be produced by bacterial strains which exhibit a beta- lactamase resistance. Beta-lactamases usually cause a hydrolytic cleavage of the beta-lactam ring of the antibiotic, so a reaction product is heavier than the antibiotic by 18 atomic mass units.
  • the determination of the inhibitory effect of a test substance can also include a quantitative determination of the inhibitory effect, i.e. the strength of the inhibitory effect.
  • the reaction speed with which a more or less inhibited beta-lactamase reacts with a substrate is a measure of the strength of the inhibitory effect of a test substance. If the concentrations of the beta-lactamases (or the quantity of biological material), the substrate and the test substance are known at the start of the method, or fixed by standardized operating instructions, the reaction speed can be determined from the concentrations of the substrate and its reaction products measured after a certain time (single-point measurement), where the concentrations are represented by the signal intensities of the relevant mass signals (or their ratios with respect to each other).
  • the reaction speed can also be determined each with from the concentrations of the substrate or from the concentrations of reaction products. But it is also possible to acquire an additional mass spectrum (two-point measurement) at the start of the reaction process, or several additional mass spectra during the course of the reaction (multi-point measurement) in order to determine the reaction speed from the temporal changes of the mass signals in the mass spectra.
  • the concentration of the substrate or the concentrations of reaction products can be derived from mass signals of calibration substances, which are added to the reaction mixture (preferably at the end of the reaction process) and cannot be enzymatically modified.
  • the inhibitory effect of a test substance on several beta-lactamases can be determined simultaneously. Since the reaction products from different substrates can usually be distinguished clearly in a mass spectrum, it is also possible to use several substrates which are each specific for a certain beta-lactamase or for a certain class of beta-lactamases so that the inhibitory efficiency of the test substance on different beta-lactamases can be distinguished.
  • the substrates used in the method can be effective beta-lactam antibiotics or derivatives of beta-lactam antibiotics. To increase the sensitivity of the method it is advantageous to use high substrate concentrations.
  • the test substance is preferably added to a nutrient solution together with several substrates and resistant bacteria.
  • the bacterial strains selected are particularly ones which have a marked beta-lactamase resistance to current beta-lactam antibiotics and combination drugs. After an incubation period, a mass spectrum is acquired from the supernatant of the nutrient solution or a portion of it.
  • the mass spectrum can be acquired with any type of mass spectrometer in the method according to the invention. It is, however, particularly advantageous to use a MALDI time-of-flight mass spectrometer with axial ion injection which is already used in routine operation for the mass spectrometric identification of microorganisms. Since the MALDI process generates a strong chemical background in the lower mass range of a few hundred atomic mass units, it is favorable to use substrates with masses in the mass ranges with low background. This is achieved, for example, by using substrates with molecular weights greater than 700 atomic mass units, preferably between 700 and 1200 atomic mass units. It is also advantageous to increase the proton affinity of the substrates in order to be easier ionized.
  • a chemical derivatization of the substrate with regard to the molecular weight, the proton affinity and/or a covalently bound charge tag is preferably carried out at the end of the reaction process in order to leave the chemical properties of the substrate as similar as possible to those of an antibiotic during the reaction, and can also be applied to reaction products of the substrate.
  • the substrates used can have an anchor group with which they can be extracted from the medium in which the reaction was prepared, and enriched. Examples of anchor groups are biotin and a 6-His tag (a chain of six histidine molecules), which are extracted with the aid of streptavidin or a chelate loaded with nickel ions respectively; the bonds here are reversible and the extracting binding partner can be bound to the walls of the reaction vessel or to magnetic beads.
  • An application of the method according of the invention consists in screening a test substance library during a pharmacological development of drugs in order to find lead structures for beta-lactamase inhibitors which can be used in combination drugs together with an antibiotic.
  • a further application consists in taking several test substances which have different inhibitory effects on different classes of beta-lactamases, and determining their inhibitory efficiency against a bacterial strain with a beta-lactamase resistance.
  • the beta-lactamases of the bacterial strain are then characterized by the inhibitory efficiency or inefficiency of the substances, particularly with regard to the encoding site of the resistance gene.
  • the resistance gene can be encoded in a plasmid or in the bacterial chromosome.
  • beta-lactamases of molecular group A are mainly plasmid encoded, but are hardly inhibited, if at all, by clavulanic acid, while beta-lactamases of molecular group B (metalloen- zymes) are chromosomally encoded and are inhibited by clavulanic acid.
  • the invention relates also to a method for determining whether a bacterial sample is beta-lactamase resistant to a combination drug, determining particularly the inhibitory efficiency of the inhibitor of a combination drug against beta-lactamases.
  • the method according to the invention comprises: bringing together the bacterial sample and the combination drug, acquiring a mass spectrum, and determining whether a beta-lactam antibiotic of the combination drug has been enzymatically modified.
  • a combination drug consists at least of one inhibitor and one beta-lactam antibiotic. The number of antibiotics or inhibitors in a combination drug can each be greater than one, however.
  • bacterial sample is to be understood in a general sense. It can be a sample which is known or suspected to comprise bacteria, or which is to be analyzed as to whether it comprises bacteria.
  • a bacterial sample can, for example, be a portion of an already isolated bacterial culture, which is harvested from an agar plate, but can also be a bacteria pellet, which is obtained directly from a body fluid such as blood, urine or CSF (cerebrospinal fluid) or from a nutrient solution such as a blood culture or culture broth.
  • Smears from mucous membranes are also deemed to be bacterial samples, as are swab samples in hospitals or in food- processing companies, and food samples themselves.
  • a bacterial sample can comprise bacteria of one or more strains. 10029)
  • the bacterial sample is incubated together with the combination drug in a nutrient solution, and afterwards the supernatant of the nutrient, solution or a portion of it is subjected to a mass spectrometric measurement.
  • a flat nutrient medium e.g. an agar plate
  • the mass spectrum is then acquired from the culture medium close to the bacterial sample.
  • the bacterial sample is brought together only with the beta-lactam antibiotic of the combination drug.
  • the additional mass spectrum provides information on whether the bacteria of the sample produce beta- lactamases which enzymatically modify the beta-lactam antibiotic of the combination drug.
  • the method according to the invention can furthermore be modified so that the bacterial sample is not brought together with the combination drug, but with the inhibitor of the combination drug and a substrate, e.g. a derivative of the beta-lactam antibiotic.
  • an enzymatic modification of the beta-lactam antibiotic can be inferred from the absence or decrease of the mass signal of the beta-lactam antibiotic, or from the presence of one or more mass signals of reaction products, or from the intensity ratio of the mass signal of the beta-lactam antibiotic to the mass signal of a reaction product, or to the mass signal of a reference substance.
  • the speed with which a beta-lactam antibiotic of a combination drug reacts is a quantitative measure for the strength of the beta- lactamase resistance of the bacteria against the combination drug, and can be determined by a single-point, two-point or multi-point measurement. As is usually the case with a functional test, the determination of the resistance can be carried out for different concentrations of the combination drug in order to determine the minimum inhibitory concentration.
  • the method according to the invention can test rapidly and reliably whether a bacterial sample has a beta-lactamase resistance and whether a combination drug is effective against existing beta-lactamases.
  • the test takes less than three hours and is thus very much faster than the routine method where the growth of the bacteria or the inhibition of growth is detected by optical means.
  • the method according to the invention can be used to functionally determine the efficiency of combination drugs against a bacterial sample with a beta-lactamase resistance.
  • the mass spectrum in the methods according to the invention can be acquired with any type of mass spectrometer, for example with a time-of-fiight mass spectrometer with orthogonal ion injection (OTOF), an ion cyclotron resonance mass spectrometer (ICR-MS), an electrostatic ion trap mass spectrometer or an RF ion trap mass spectrometer.
  • OTOF time-of-fiight mass spectrometer with orthogonal ion injection
  • ICR-MS ion cyclotron resonance mass spectrometer
  • electrostatic ion trap mass spectrometer electrostatic ion trap mass spectrometer
  • RF ion trap mass spectrometer RF ion trap mass spectrometer
  • MALDI time-of-flight mass spectrometer with axial ion injection which is already used in routine operation for the taxonomic identification of the bacteria.
  • MALDI matrix-assisted laser desorption
  • ESI electrospray ionization
  • NIMS matrix-free laser desorption
  • Figures 1A to 1 C show the molecular structural formula of three beta-lactamase inhibitors known from the prior art: clavulanic acid (Fig. 1A), sulbactam (Fig. IB) and tazobac- tam (Fig. 1 C).
  • Figure 2 is a schematic representation of the procedure for determining the inhibitory efficiency of test substances in order to identify lead structures in the search for pharmacologically active substances (high-throughput screening).
  • Figure 3 is a schematic representation of the procedure for determining the susceptibility of a combination drug against a bacterial sample.
  • Figures 4A to 4D show four measured MALDI mass spectra of the ampicillin antibiotic in a mass range between 340 and 450 atomic mass units. Examples of Preferred Embodiments
  • Figures 1A to 1C show the molecular structural formula of three beta-lactamase inhibitors known from the prior art: clavulanic acid (Fig. 1 A), sulbactam (Fig. IB) and tazobac- tam (Fig. 1C). These inhibitors all have a beta-lactam ring, i.e. a cyclic amide with a heterocyclic structure of three carbon atoms and one nitrogen atom.
  • Figure 2 is a schematic representation of the procedure for determining the inhibitory efficiency of test substances in order to identify lead structure elements in the search for pharmacologically active substances. Pharmaceutical companies keep large substance libraries which can easily contain a few hundred thousand different substances.
  • the method according to the invention makes it possible to test a large number of test substances in a short time for their inhibitory efficiency against beta-lactam ases (high-throughput screening).
  • test substances are provided in a substance plate 100.
  • the substance plate 100 is a microtiter plate with 24 spatially separate wells 101 to 124, which are arranged in rows and columns.
  • the precise dimensions (length x width x height) of a microtiter plate according to the ANSI standard, on the recommendation of the Society of Biomolecular Screening, are 127.76 mm x 85.48 mm x 14.35 mm.
  • There are a multitude of formats e.g. with 24 wells (4x6, capacity: 0.5-3 ml per well), 96 wells (8 12, capacity: 0.3-2 ml per well) or 384 wells (16x24, capacity: 0.03-0.1 ml per well).
  • Each well on the substance plate 100 contains only one single test substance in this embodiment.
  • a pipetting robot 10 transfers the test substances from the substance plate 100 onto a reaction plate 200 in such a way that each well on the reaction plate
  • the reaction plate 200 contains only one test substance.
  • the reaction plate 200 is a microtiter plate with 24 wells
  • Step B a substrate mixture with three different substrates SI to S3, which are all beta-lactam antibiotics, is put into each well of the reaction plate 200 by means of a dispenser 20.
  • the dispensing is a non-contact operation in order to prevent contamination between the reaction mixtures.
  • Step C a few microliters of the supernatant from each well of the reaction plate 200 is transferred onto a first MALDI sample support and prepared there.
  • the sample support 300 has the same format as the reaction plate 200 with respect to number and positions of the sample sites, but can also have more than the 24 sample sites 301 to 324 so that samples from other reaction plates can also be prepared on the MALDI sample support 300.
  • the matrix used is a-cyano-4- hydroxycinnamic acid (HCCA) at a concentration of 10 milligrams per milliliter in a mixture of water, 50% acetonitrile and 2.5% trifmoroacetic acid.
  • a pipetting robot (not shown) transfers the samples and the matrix solution onto the sample sites 301 to 324 of the MALDI sample support 300.
  • the sample support 300 is introduced into a MALDI time-of- flight mass spectrometer and mass spectra MSal to MSa24, of which only the mass spectra MSal and MSa24 are shown in Figure 3 for reasons of clarity, are acquired at each of the MALDI sample sites 301 to 324.
  • Each of the MALDI mass spectra MSal to MSa24 comprises mass signals of the substrates S I to S3 and of the relevant test substance, for example the test substance Tl in the mass spectrum MSal .
  • the mass spectra MSal to MSa24 acquired in Step C represent the signal strengths of the substrates SI to S3 at the start of the subsequent inhibition reactions and thus allow the determination of the inhibitory strength of the test substances under investigation Tl to T24 in a two-point measurement.
  • Step D a bacterial mixture is put into each well of the reaction plate 200 by means of a dispenser 30.
  • the dispensing is a non-contact operation, as in Step C, in order to prevent contamination between the reaction mixtures.
  • the bacterial mixture contains different bacterial strains for which a beta-lactamase resistance to the antibiotics used has already been proven.
  • the reaction plate 300 is subsequently incubated for three hours at 37° Celsius under constant shaking and then centrifuged in order to precipitate the bacterial cells at the bottom of the wells.
  • Step E a few microliters of the supernatant are transferred from each well of the reaction plate 200 onto a second MALDI sample support and prepared there as described in Step C,
  • the sample support 400 has the same format as the sample support 300.
  • the sample support 400 is introduced into the MALDI time-of-flight mass spectrometer and one mass spectrum is acquired for each of the 24 MALDI sample sites 401 to 424 (MSbl to MSb24).
  • the inhibitory efficiency of the test substances can be determined by com- paring the corresponding mass signals in the mass spectra before and after the inhibition reaction.
  • the mass signals of the substrates SI to S3, for example, in the mass spectrum MSbl are unchanged in comparison to the mass spectrum MSal , i.e.
  • test substance Tl inhibits all the beta-lactamases produced by the bacterial mixture.
  • mass spectrum MSb24 in contrast, the mass signals of the substrates SI to S3 are missing, and mass signals of corresponding reaction products Rl to R3 are present.
  • the test substance T24 does not inhibit the beta-lactamases of the bacterial mixture.
  • the method can be carried out in such a way that the inhibitory effect is at first determined in a large number of multiplex mix- tures, each with several test substances per well, and that subsequently those test substances which exhibited an inhibitory effect in one of the multiplex mixtures are tested individually.
  • Figure 3 is a schematic representation of a procedure for determining the susceptibility of a combination drug against a bacterial sample.
  • Step A small quantities of an isolated bacterial colony 3, which was cultured in the usual way on an agar plate, are each transferred into two Eppendorf tubes 1 and 2, which are then filled with a liquid nutrient medium. The Eppendorf tubes 1 and 2 are then vortexed for a short time to distribute the bacteria in the nutrient medium. To standardize the test, the number of bacterial cells per unit volume is adjusted to a specific value by the addition of further quantities of microbes, or by an incubated growth phase, or by dilution of the nutrient medium, until the optical density of the nutrient medium reaches a certain value.
  • Step B the combination drug under investigation 5 is added to the reaction mixture in the Eppendorf tube 1. Only the antibiotic 6 of the combination drug is added to the reaction mixture in the Eppendorf tube 2.
  • Steps C and D the two Eppendorf tubes 1 and 2 are incubated and shaked at 37°C for three hours and subsequently centrifuged for 2 minutes at 13,000 rpm in order to obtain a cell-free supernatant (7A, 8 A) and a bacterial pellet ( 7B. 8B) at the bottom of both Eppendorf tubes 1 and 2.
  • Step E one microliter from the supernatant 7A and one microliter from the supernatant 8A are transferred to two separate sample sites of a MALDI sample support. After the samples transferred from the supernatant have dried, they are coated with one microliter of a matrix solution.
  • the matrix solution used is a-cyano-4-hydroxycinnamic acid (HCCA) at a concentration of 10 milligrams per milliliter in a mixture of water, 50% acetonitrile and 2.5% trifluoroacetic acid.
  • HCCA a-cyano-4-hydroxycinnamic acid
  • the sample support is introduced into a MALDI time-of-flight mass spectrometer and MALDI mass spectra MSI and MS2 are acquired in the mass range from 100 to 1000 atomic mass units.
  • MALDI time-of-flight mass spectrometer After the matrix has crystallized out by vaporization of the solvent, the sample support is introduced into a MALDI time-of-flight mass spectrometer and MALDI mass spectra MSI and MS2 are acquired in the mass range from 100 to 1000 atomic mass units.
  • Other matrices, solvents and types of MALDI preparation are possible.
  • the mass spectrum MS2 exhibits a mass signal of the antibiotic A and a reaction product R, i.e. that the antibiotic A has been enzymatically modified and the bacterial colony 3 comprises a beta-lactamase resistance.
  • the mass spectrum MSI exhibits only the mass signal of the antibiotic A, but no mass signals of reaction products, i.e. the inhibitor of the combination drug inhibits the beta-lactamases formed by the bacterial colony, and the combination drug can be used for a therapy against this bacterial strain.
  • Figures 4A to 4D show four measured MALDI mass spectra with the ampicillin antibiotic in a mass range between 340 and 450 atomic mass units.
  • Ampicillin has a molar mass of 349.41 g/mol and is a semi-synthetic, antibiotic active substance from the group of the beta-lactam antibiotics, which has been used since 1961. It is known as a broad-spectrum antibiotic due to its efficiency against Gram-positive bacteria and some Gram-negative bacterial species. Ampicillin belongs chemically to the aminopenicillins.
  • the sample preparation (inoculation of a liquid nutrient medium, incubation and centrifugation) and the preparation of the MALDI samples correspond to the method in Figure 3.
  • the mass spectrum in Figure 4A shows an ampicillin spectrum after incubation with the DH5a bacteria strain of the species Escherichia coli.
  • the mass spectrum exhibits the mass signals of the protonated ampicillin ([M+H] + , 350u) and of a sodium adduct ion of the ampicillin ([M+Na] + , 372u), but no mass signals of reaction products which could be attributed to an enzymatic modification, such as a hydrolytic cleaving of the beta-lactam ring.
  • the bacterial strain DH5a does not exhibit any beta-lactamase resistance.
  • the mass spectrum in Figure 4B depicts an ampicillin spectrum after incubation with an ESBL bacterial strain of the species Escherichia coli.
  • the two mass signals of the ampicillin at 350u and 372u are absent, and hydrolyzed reaction products are present in the mass spectrum at 368u and 390u (18u heavier).
  • the ESBL bacterial strain produces beta-lactamases which enzymatically modify the ampicillin and it is therefore resistant to ampicillin.

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Abstract

L'invention concerne un procédé de détermination de l'effet inhibiteur d'une substance sur des bêta-lactamases, selon lequel la substance est mélangée avec une bêta-lactamase et un substrat qui contient un cycle bêta-lactame et, après un temps de réaction donné, un spectre de masse est acquis et évalué pour déterminer si le substrat a été modifié enzymatiquement par les bêta-lactamases.
PCT/EP2012/052662 2011-02-23 2012-02-16 Détermination par spectrométrie de masse de l'effet inhibiteur de substances sur les bêta-lactamases WO2012113699A1 (fr)

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DE201110012060 DE102011012060A1 (de) 2011-02-23 2011-02-23 Massenspektrometrische Bestimmung der inhibitorischen Wirkung von Substanzen gegenüber beta-Lactamasen
DE102011012060.2 2011-02-23

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WO2016016580A1 (fr) * 2014-07-30 2016-02-04 bioMérieux Caracterisation de micro-organismes par maldi-tof
WO2019098643A1 (fr) * 2017-11-14 2019-05-23 주식회사 노스퀘스트 Appareil et procédé pour déterminer une résistance aux antibiotiques par spectrométrie de masse maldi-tof
CN111254185A (zh) * 2013-05-23 2020-06-09 布鲁克道尔顿有限公司 通过生长测量的质谱法抗药性测定
KR20210084040A (ko) 2019-12-27 2021-07-07 주식회사 노스퀘스트 Maldi-tof를 이용한 카바페넴 분해효소-생성 장내세균 검출
US11142784B2 (en) 2014-01-17 2021-10-12 Bruker Daltonik Gmbh Mass spectrometric resistance determination by measuring metabolism
CN111254185B (zh) * 2013-05-23 2024-05-31 布鲁克·道尔顿有限及两合公司 通过生长测量的质谱法抗药性测定

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CN111254185A (zh) * 2013-05-23 2020-06-09 布鲁克道尔顿有限公司 通过生长测量的质谱法抗药性测定
WO2014202034A1 (fr) * 2013-06-20 2014-12-24 Univerzita Karlova V Praze, Lekarska Fakulta V Plzni Procédé de détection de protéines de l'espace périplasmique et de la membrane externe de la paroi cellulaire de bactéries à gram négatif par spectrométrie de masse
EP2816357A1 (fr) * 2013-06-20 2014-12-24 Univerzita Karlova V Praze Procédé de détection d'espace périplasmique à bactéries gram négatives et protéines de membrane externe de paroi cellulaire par spectrométrie de masse
US20160138068A1 (en) * 2013-06-20 2016-05-19 Univerzita Karlova V Praze, Lekarska Fakulta V Plzni Method of detection of gram-negative bacteria periplasmic space and cell wall outer membrane proteins by mass spectrometry
US9803229B2 (en) * 2013-06-20 2017-10-31 Univerzita Karlova V Praze, Lekarska Fakulta V Plzni Method of detection of gram-negative bacteria periplasmic space and cell wall outer membrane proteins by mass spectrometry
US11142784B2 (en) 2014-01-17 2021-10-12 Bruker Daltonik Gmbh Mass spectrometric resistance determination by measuring metabolism
EP3495492A1 (fr) * 2014-07-30 2019-06-12 bioMérieux Caracterisation de micro-organismes par maldi-tof
RU2698139C2 (ru) * 2014-07-30 2019-08-22 Биомерьё Описание характеристик микроорганизмов с помощью maldi-tof
JP2017523420A (ja) * 2014-07-30 2017-08-17 ビオメリューＢｉｏｍｅｒｉｅｕｘ Ｍａｌｄｉ−ｔｏｆを介した微生物の特性解析
JP2020122794A (ja) * 2014-07-30 2020-08-13 ビオメリューＢｉｏｍｅｒｉｅｕｘ Ｍａｌｄｉ−ｔｏｆを介した微生物の特性解析
FR3024465A1 (fr) * 2014-07-30 2016-02-05 Biomerieux Sa Caracterisation de micro-organismes par maldi-tof
JP6992112B2 (ja) 2014-07-30 2022-01-13 ビオメリュー Ｍａｌｄｉ－ｔｏｆを介した微生物の特性解析
WO2016016580A1 (fr) * 2014-07-30 2016-02-04 bioMérieux Caracterisation de micro-organismes par maldi-tof
WO2019098643A1 (fr) * 2017-11-14 2019-05-23 주식회사 노스퀘스트 Appareil et procédé pour déterminer une résistance aux antibiotiques par spectrométrie de masse maldi-tof
CN111344572A (zh) * 2017-11-14 2020-06-26 株式会社诺斯凯斯特 基于maldi-tof质量分析的抗生素抗性辨别装置及方法
KR20210084040A (ko) 2019-12-27 2021-07-07 주식회사 노스퀘스트 Maldi-tof를 이용한 카바페넴 분해효소-생성 장내세균 검출

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