Mass spectrometric measurement of microbial resistances
View Patent ↗Microorganisms, particularly bacteria, are identified and characterized on the basis of a mass spectrometric measurement of their protein profiles with ionization by matrix-assisted laser desorption. In order to measure the microbial resistance to antibiotics, the protein profiles of microorganisms are measured after cultivation for a short time duration in nutrient media containing the antibiotics.
1. A method for determining the resistance of microbes to a specific antibiotic, comprising:
(a) adding a quantity of the microbes to a nutrient medium that also contains a quantity of the antibiotic and adding to the nutrient medium reactive substances that can reactively modify microbes which are weakened by the antibiotic;
(b) incubating the nutrient medium at a predetermined temperature for a predetermined time;
(c) removing the microbes from the incubated nutrient medium;
(d) applying the removed microbes and a matrix solution to a mass spectrometric sample support and drying the matrix solution on the sample support to produce a sample;
(e) acquiring a mass spectrum of the sample; and
(f) determining resistance of the microbes to the antibiotic by measuring modifications of the mass spectrum effected by the antibiotic compared to at least one reference mass spectrum of the microbes.
2. The method of claim 1 , wherein step (a) comprises adding a quantity of the microbes to a liquid nutrient medium.
3. The method of claim 2 , wherein the liquid nutrient medium is contained in a microvessel of a microtitration plate.
4. The method of claim 1 , wherein the reactive substances are enzymes.
5. The method of claim 1 , wherein step (c) comprises removing the microbes from the incubated nutrient medium by filtration or centrifuging.
6. The method of claim 1 , wherein step (d) comprises applying the removed microbes to the mass spectrometric support and sprinkling the matrix solution onto the microbes on the support.
7. The method of claim 1 , wherein step (d) comprises mixing the matrix solution with the removed microbes, applying the matrix solution and microbe mixture to the mass spectrometric sample support and drying the mixture on the sample support to produce the sample.
8. The method of claim 1 , wherein, in step (a) the quantity of microbes is taken from a homogeneous colony.
9. The method of claim 8 , wherein, in step (f), the reference mass spectrum is a mass spectrum of microbes taken from the homogeneous colony.
10. The method of claim 1 , wherein, in step (f), the reference mass spectrum is a mass spectrum of microbes cultivated in the same way as the microbes but without being subjected to effect of the antibiotic.
11. The method of claim 1 , wherein, in step (f), the reference mass spectrum is a mass spectrum of reference microbes in a resistant form acquired after the antibiotic has acted on the reference microbes.
12. The method of claim 11 , wherein the reference mass spectrum is contained in a spectral library which also contains mass spectra of the reference microbes.
13. The method of claim 1 , wherein in step (f), the reference mass spectrum is a mass spectrum of reference microbes in a non-resistant form acquired after the antibiotic has acted on the reference microbes.
14. The method of claim 13 , wherein the reference mass spectrum is contained in a spectral library which also contains mass spectra of the reference microbes.
15. A method for determining the resistance of microbes to a specific antibiotic, comprising:
(a) adding a quantity of the microbes to a nutrient medium that also contains a quantity of the antibiotic and adding to the nutrient medium, marked nutrients whose intake by the microbes can be detected mass spectrometrically;
(b) incubating the nutrient medium at a predetermined temperature for a predetermined time;
(c) removing the microbes from the incubated nutrient medium;
(d) applying the removed microbes and a matrix solution to a mass spectrometric sample support and drying the matrix solution on the sample support to produce a sample;
(e) acquiring a mass spectrum of the sample; and
(f) determining resistance of the microbes to the antibiotic by measuring modifications of the mass spectrum effected by the antibiotic compared to at least one reference mass spectrum of the microbes.
16. The method of claim 15 , wherein the marked nutrients are substances marked with isotopes.
17. The method of claim 15 , wherein step (a) comprises adding reference microbes in addition to the microbes to the nutrient medium.
18. The method of claim 17 , wherein the reference microbes are selected to quantitatively evaluate the growth of the microbes.
19. A method for determining the resistance of microbes to a specific antibiotic, comprising:
(a) adding a quantity of the microbes to a nutrient medium that also contains a quantity of the antibiotic and a marked nutrient whose intake by the microbes can be detected mass spectrometrically;
(b) incubating the microbes in the nutrient medium at a predetermined temperature for a predetermined time;
(c) removing the microbes from the nutrient medium;
(d) applying the removed microbes and a matrix solution to a mass spectrometric sample support and drying the matrix solution on the sample support to produce a sample;
(e) acquiring a mass spectrum of the sample; and
(f) determining the microbes to be resistant to the antibiotic if the marked nutrient is detected in the mass spectrum.