Methods for screening compounds for bactericidal activity and for determining the sensitivity of bacterial samples
AssigneesCentre National de la Recherche Scientifique (CNRS), Universite Paris-Saclay
InventorsDominique Fourmy, Satoko Yoshizawa, Marie Ebeyer-Masotta, Jean-Luc Pernodet
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Abstract
The present invention relates to a method for screening compounds for bactericidal activity using thermostable luciferase and based on a real-time bioluminescence measurement. The present invention further relates to a method for determining the sensitivity of a bacterial sample originating from a subject suffering from a bacterial infection to a group of known antibiotics and to a method for assessing the minimum inhibitory concentration (MIC) of a bactericidal compound.
Claims20
A method for screening compounds for bactericidal activity, comprising:
- a) providing one or more test bacterial sample(s) comprising live bacteria in a culture medium;
- b) adding a mixture of luciferin and a thermostable luciferase to said test bacterial sample(s);
- c) adding a candidate composition to said test bacterial sample(s) 2 to 15 min after step b);
- and
- d) incubating the test bacterial sample(s) to which the mixture of luciferin and thermostable luciferase and the candidate composition have been added at a temperature between 20 and 60° C., and measuring bioluminescence in real-time;
- wherein the optical density at 600 nm (OD600) of live bacteria in the test bacterial sample(s) after steps a), b) and c) have been performed is at least 0.0002, and
- wherein an increase of bioluminescence measured in a test bacterial sample in step d) is indicative that the candidate composition added to the test bacterial sample in step c) comprises at least one compound with bactericidal activity.
A method for determining the sensitivity of a bacterial sample originating from a subject suffering from a bacterial infection to a group of known antibiotics, comprising:
- a) inoculating a bacterial sample originating from a subject suffering from a bacterial infection into a culture medium;
- b) dividing the bacterial sample of step a) into several sub-samples, with at least as many samples as the number of known antibiotics to be tested;
- c) adding a mixture of luciferin and a thermostable luciferase to each sub-sample;
- d) adding each of the group of known antibiotics to one or more sub-sample(s);
- 2 to 15 min after step c);
- and
- e) incubating the sub-sample(s) to which the mixture of luciferin and thermostable luciferase and the known antibiotic have been added at a temperature between 20 and 60° C. and measuring bioluminescence in real-time;
- wherein the optical density at 600 nm (OD600) of live bacteria in the sub-samples after steps a), b), c) and d) have been performed is at least 0.0002, and
- wherein an increase of bioluminescence measured in a sub-sample in step e) is indicative that the bacterial sample originating from a subject is sensitive to the added concentration of known antibiotic added to the sub-sample in step d).
A method for assessing the minimum inhibitory concentration (MIC) of a bactericidal compound comprising:
- a) providing at least one test bacterial sample comprising live bacteria in a culture medium;
- b) dividing the bacterial sample of step a) into several sub-samples,
- c) adding a mixture of luciferin and a thermostable luciferase to said sub-samples;
- d) adding varying concentrations of a bactericidal compound to said sub-samples 2 to 15 min after step c);
- e) incubating the sub-samples to which the mixture of luciferin and thermostable luciferase and the bactericidal compound have been added at a temperature between 20 and 60° C. and measuring bioluminescence in real-time, wherein the optical density at 600 nm (OD600) of live bacteria in the sub-samples after steps a), b), c) and d) have been performed is at least 0.0002;
- f) determining for each concentration of bactericidal compound tested the lag time between the time when said bactericidal compound has been added and the time of detection of an increase in the bioluminescence signal,
- g) representing the lag time in function of the bactericidal compound concentration,
- h) creating an exponential decay curve fitting the measured points of the lag time in function of the bactericidal compound concentration,
- i) determining the lowest bactericidal compound concentration in the plateau of the exponential decay fitting curve,
- j) determining the lag time amplitude of the exponential decay fitting curve, and
- k) assessing the MIC, wherein the MIC is assessed as comprised between:
- the antibiotic concentration corresponding on the exponential decay fitting curve to a lag time equal to (lag time at plateau +0.3× lag time amplitude), and
- the lowest bactericidal compound concentration in the plateau of the exponential decay fitting curve.
Dependencies are inferred from the claim text.