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) cultivating a first quantity of the microbes in a nutrient medium containing the antibiotic;
(b) cultivating a second quantity of the microbes using the same nutrient medium and cultivation conditions used in step (a) except that the nutrient medium does not contain the antibiotic;
(c) preparing a first sample from the microbes cultivated in step (a) using whole microbes or proteins extracted from the microbes;
(d) preparing a second sample from the microbes cultivated in step (b) using whole microbes or proteins extracted from the microbes;
(e) measuring a first mass spectrum of the first sample that is indicative of the soluble proteins of the microbes cultivated in step (a);
(f) measuring a second mass spectrum of the second sample that is indicative of the soluble proteins of the microbes cultivated in step (b);
(g) comparing the first and second mass spectra; and
(h) determining the resistance of the microbes based on the comparison of step (g), wherein similarity of the spectra indicates resistance.
2. The method of claim 1 , wherein the nutrient medium is 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 2 , further comprising removing the first and second samples of the microbes from the nutrient medium by filtration or centrifuging.
5. The method of claim 1 , wherein nutrient medium used in step (a) contains reactive substances that can reactively modify microbes which are weakened by the antibiotic.
6. The method of claim 5 , wherein the reactive substances are enzymes.
7. The method of claim 1 , wherein the nutrient medium used in step (a) and in step (b) contains marked nutrients whose intake by the microbes can be detected mass spectrometrically.
8. The method of claim 7 wherein the marked nutrients are substances marked with isotopes.
9. A method for determining the resistance of microbes having a known mass spectrum to a specific antibiotic, comprising:
(a) adding a quantity of the microbes and a quantity of reference microbes to a single nutrient medium that contains a quantity of the antibiotic, the reference microbes having a known mass spectrum and being of a type similar to the microbes but resistant to the antibiotic;
(b) incubating the microbes and reference microbes in the single nutrient medium at a predetermined temperature for a predetermined time;
(c) removing the microbes and the reference microbes from the single nutrient medium;
(d) applying the removed microbes, the removed reference 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 acquired mass spectrum is substantially a superposition of the known mass spectrum of the microbes and the known mass spectrum of the reference microbes.