Granted Patent B2

US 12,435,352 · App. 13/582,372 · Granted Oct 7, 2025

Inventors: Markus Kostrzewa (Lilienthal, DE); Karsten Michelmann (Bremen, DE); Katrin Sparbier (Bremen, DE)

Assignee: Bruker Daltonics GmbH & Co. KG

C12Q1/18C12Q1/34G01N33/6851G01N2333/986G01N2800/26G01N2800/44

Abstract

Claims (25)

1. A method for the determination of a β-lactam resistance of microbes based on the production of β-lactamases by the microbes, the method comprising:

obtaining the microbes from a colony cultivated on a nutrient medium or from a blood culture;

combining the microbes with a β-lactam antibiotic covalently bonded to a 6-His tag in a solution, wherein β-lactamases of the microbes may cause hydrolytic breakdown of the β-lactam antibiotic;

extracting remaining 3-lactam antibiotic and/or its breakdown product from the solution via a reversible bond of the 6-His tag to an extracting partner;

partly applying the extracted remaining-lactam antibiotic and/or breakdown product to a mass spectrometric support; and

measuring an enzymatic breakdown of the β-lactam antibiotic by the β-lactamases of the microbes mass spectrometrically by acquiring a mass spectrum of the extracted remaining β-lactam antibiotic and/or breakdown product using a time of flight mass spectrometer with ionization by matrix-assisted laser desorption, wherein a matrix used is α-cyano-4-hydroxycinnamic acid.

2. The method according to claim 1 , wherein the β-lactam antibiotic has a molecular weight between 700 and 1200 atomic mass units.

3. The method according to claim 1 , wherein the microbes are combined with different types of β-lactam antibiotics in a single multiplex resistance assay and the breakdown of the different types of β-lactam antibiotics is measured simultaneously.

4. The method according to claim 3 , wherein the different types of β-lactam antibiotics are customized such that their breakdown pattern makes it possible to identify the different classes of β-lactamases.

5. The method according to claim 4 , wherein the different types of β-lactam antibiotics are derivatized with different chemical groups around the β-lactam antibiotic to imitate the steric forms of different antibiotics.

6. The method according to claim 1 , wherein the β-lactam antibiotic is one of penicillins, benzylpenicillins, oral penicillins, aminopenicillins, isoxazolylpenicillins, acylaminopenicillins, cephalosporins, monobactams and carbapenems.

7. The method according to claim 1 , wherein the β-lactam antibiotic is ampicillin combined with sulbactam, amoxicillin combined with clavulanic acid or piperacillin combined with tazobactam.

8. The method according to claim 1 , wherein the concentration of the β-lactam antibiotic in the solution is dosed such that the microbes will not survive and that mass spectrometric signals of the antibiotic and the breakdown product are recognizable above a chemical background.

9. A method for the determination of a β-lactam resistance of microbes based on the production of β-lactamases by the microbes, the method comprising:

obtaining the microbes from a colony cultivated on a nutrient medium or from a blood culture;

combining the microbes with a β-lactam antibiotic covalently bonded to a 6-His tag in a solution, wherein β-lactamases of the microbes may cause hydrolytic breakdown of the β-lactam antibiotic;

extracting remaining-lactam antibiotic and/or its breakdown product from the solution via a reversible bond of the 6-His tag to an extracting partner;

partly applying the extracted remaining β-lactam antibiotic and/or breakdown product to a mass spectrometric support; and

measuring the enzymatic breakdown of the β-lactam antibiotic by the β-lactamases of the microbes mass spectrometrically by acquiring a mass spectrum of the extracted remaining β-lactam antibiotic and/or breakdown product using ionization by matrix-assisted laser desorption, wherein a matrix used is α-cyano-4-hydroxycinnamic acid.

10. The method according to claim 9 , wherein the concentration of the β-lactam antibiotic in the solution is dosed such that the microbes will not survive and that mass spectrometric signals of the antibiotic and the breakdown product are recognizable above a chemical background.

11. A method for the determination of a β-lactam resistance of microbes based on the production of β-lactamases by the microbes, the method comprising:

obtaining the microbes from a colony cultivated on a nutrient medium or from a blood culture;

combining the microbes with a substrate in a solution, said substrate comprising a β-lactam ring;

partly applying the solution to a mass spectrometric support; and

measuring an enzymatic breakdown of the substrate by the β-lactamases of the microbes mass spectrometrically by acquiring a mass spectrum of remaining substrate and breakdown product, which are embedded in crystals of a matrix substance, using a time of flight mass spectrometer with ionization by matrix-assisted laser desorption, wherein the breakdown product comprises a mass which is 18 atomic mass units heavier than the substrate and wherein the matrix used is α-cyano-4-hydroxycinnamic acid.

Assignments (2)

Priority Claims (1)

Continuity (1)

References Cited (50)