Rapid test for microbial resistances by mass spectrometry
View Patent ↗The invention relates to methods and instruments for determining the resistances of microbes to antibiotics, in particular those microbes which produce beta-lactamases. The method determines the resistance of the microbes in less than an hour by incubating a tiny quantity of the microbes on a mass spectrometric sample support plate after they have been combined with a dosed quantity of a suitable antibiotic, for example the beta-lactam antibiotic imipenem, and by direct mass spectrometric measurement of the breakdown of the antibiotic by the microbial enzymes.
1. A method for determining the resistance of a microbial sample to an antibiotic, the method comprising:
providing a flat sample support plate having a test site coated with a dosed quantity of the antibiotic and with one or more reference substances;
combining the microbial sample and the antibiotic in a volume of liquid on the test site to form a measurement sample; and
analyzing the measurement sample by a mass spectrometric measurement to determine whether the antibiotic is enzymatically modified.
2. The method according to claim 1 , wherein the microbial sample comprises intact microbial cells.
3. The method according to claim 2 , further comprising incubating the microbial sample and the antibiotic in the volume of liquid at the test site for less than an hour.
4. The method according to claim 3 , wherein the incubation in the volume of liquid is carried out at room temperature.
5. The method according to claim 2 , wherein between 10 3 and 10 7 microbial cells are applied to the test site.
6. The method according to claim 2 , wherein the measurement sample is prepared at the test site for matrix-assisted laser desorption and ionization without the microbes being digested.
7. The method according to claim 2 , further comprising:
applying the intact microbial cells and the liquid onto the layer of the antibiotic and one or more reference substances,
incubating the microbial cells on the test site,
drying the measurement sample,
preparing the measurement sample for matrix-assisted laser desorption and ionization (MALDI),
acquiring a mass spectrum of the measurement sample in a mass spectrometer with a MALDI ion source, and
evaluating the mass spectrum to identify said resistance.
8. The method according to claim 1 , wherein the layer on the test site contains around 0.1 to 2 micrograms of antibiotic.
9. The method according to claim 1 , wherein the measurement sample is prepared for matrix-assisted laser desorption and ionization at the test site, whereby the microbes in the microbial sample are lysed, and mass signals of microbial proteins are acquired in the mass spectrometric measurement, said mass signals allowing the microbes to be identified taxonomically and/or additionally characterized with respect to their resistance behavior.
10. The method according to claim 1 , wherein the microbial sample comprises lysed microbes.
11. The method according to claim 1 , wherein the volume of liquid of the measurement sample is maintained for a specified reaction time by at least one of locating the measurement sample in a humid environment and adding additional liquid to the volume of liquid on the test site after said combining.
12. The method according to claim 1 , wherein said volume of liquid is less than ten microliters.
13. The method according to claim 12 , wherein said volume of liquid is between one and three microliters.
14. The method according to claim 1 , wherein the test site is coated with multiple antibiotics and the microbial sample is combined with said multiple antibiotics in the volume of liquid at the test site.
15. The method according to claim 1 , wherein the antibiotic is a beta-lactam antibiotic.
16. The method according to claim 15 , wherein the beta-lactam antibiotic is selected from the groups of penicillins, the cephalosporins, the monobactams or the carbapenems.
17. The method according to claim 15 , wherein the beta-lactam antibiotic is selected from the groups of benzylpenicillins, oral penicillins, aminopenicillins, isoxacylpenicillins, or acylaminopenicillins.
18. The method according to claim 15 , wherein the antibiotic comprises a beta-lactam antibiotic in combination with a beta-lactamase inhibitor.
19. The method according to claim 18 , wherein the beta-lactamase inhibitor is sulbactam.
20. The method according to claim 18 , wherein the antibiotic comprises amoxicillin in combination with clavulanic acid, piperacillin in combination with tazobactam or ampicillin in combination with sulbactam.
21. The method according to claim 1 , wherein the measurement sample is prepared for matrix-assisted laser desorption and ionization (MALDI) for the mass spectrometric measurement.
22. The method according to claim 1 , wherein the sample support comprises between 48 and 386 test sites.
23. The method according to claim 1 , wherein the test site is hydrophilic with hydrophobic surroundings.