Method for screening compounds for bactericidal activity and determining susceptibility of bacterial samples - Patent Application 20070122997

Abstract

The present invention relates to a method for screening compounds for bactericidal activity based on real-time bioluminescence measurements using thermostable luciferase. The present invention further relates to a method for determining the susceptibility of bacterial samples from subjects suffering from bacterial infections to a group of known antibiotics, and to a method for assessing the minimum inhibitory concentration (MIC) of bactericidal compounds.

Description

[Technical Field]

[0001] The present invention relates to an easy, simple, reliable and very rapid method for screening compounds for bactericidal activity. The present invention also relates to an equally easy, simple, reliable and very rapid method for determining the susceptibility of bacterial samples from subjects suffering from bacterial infections to a group of known antibiotics. Finally, the present invention relates to a rapid method for assessing the minimum inhibitory concentration (MIC) of bactericidal compounds. [Background technology]

[0002] In antimicrobial drug discovery and susceptibility testing, the ability to analyze vast libraries of compounds and solutions from pharmaceutical manufacturers in real time is essential. Furthermore, in treating patients with bacterial infections, the rapid determination of available bactericidal compounds and their appropriate minimum inhibitory concentrations (MINCs) is crucial to contain the infection. In situations such as an unprecedented bacterial infection pandemic, rapid screening of effective drugs is crucial. However, current tests for screening for new potent bactericidal compounds or testing the susceptibility of bacterial samples to known antibiotics are unsatisfactory. In particular, most are cumbersome and require many hours to produce results. Furthermore, most known assays can only be performed using growing bacteria, not stationary-phase or biofilm bacteria. When testing large numbers of patients, antibiotic susceptibility testing must be simple, rapid, and provide reliable results. For example, Ling et al. (Nature. 2015 Jan 22;517(7535):455-9) tested extracts from 10,000 bacterial isolates for their ability to inhibit bacterial growth using a method involving a 20-hour incubation step. This growth inhibition assay also involves growing bacteria on agar plates to form a lawn. Therefore, this assay is very time-consuming and cannot be performed on cells in the stationary phase of growth or within a biofilm. Furthermore, most antibiotic susceptibility testing relies on time-consuming bacterial growth inhibition assays.

[0003] Adenosine-5'-triphosphate (ATP) is an important biomolecule, and its quantitative detection is the subject of significant development, particularly in the fields of antimicrobial drug discovery and susceptibility testing. Indeed, the disruption of the cell membrane that accompanies cell lysis is characterized by the release of intracellular ATP stocks. Therefore, some researchers have proposed that it should be possible to measure bacterial cell lysis by bactericidal compounds by measuring the amount of ATP released into the cell culture medium. This has been attempted by two groups. In the 1990s, de Rautlin de la Roy et al. (J. Biolumin. Chemilumin. 6, 193-201 (1991)) attempted to measure the kinetics of antibiotic bactericidal activity using a luciferin-luciferase assay. In this test, the bactericidal effect of antibiotics was detected only gradually, sometimes only after 10 hours of addition to the bacterial sample. Therefore, this test is not suitable for rapid and efficient screening of novel potent bactericidal compounds. Furthermore, the dilution of bacteria into distilled water subjects the bacteria to an osmotic shock, which is expected to alter the bacterial membrane and lead to ATP release by factors other than the antibiotic, making the results unclear and making this test unreliable.

[0004] Recently, Heller et al. (2019. PLoS ONE 14(1): 1-13 (2019)) described a bacterial susceptibility test based on the ATP / OD600 ratio method, which involves measuring ATP released by cell lysis using a bioluminescent luciferin-luciferase reaction. In this method, the authors recommend adding antibiotics only when the growth phase is nearly complete and the bacteria have reached stationary phase. The use of stationary-phase cells is due to the low sensitivity of this method. The time required to reach stationary phase makes this proposed method too time-consuming. Furthermore, international antibiotic susceptibility testing guidelines discourage the use of stationary-phase cells because some antibiotics, such as aminoglycosides, are less effective against stationary-phase cells. The authors also recommend adjusting the measurement of extracellular ATP concentration based on the optical density (OD) of the culture, which may compromise the reliability of the test. Furthermore, in this test, the test sample is left on ice for a while before measuring bioluminescence. This step may induce decomposition of ATP present in the test sample, which also affects the reliability and sensitivity of the test. In fact, this method produces very weak signals and even fails to detect the susceptibility of susceptible bacteria to the aminoglycoside gentamicin. Furthermore, before measuring the ATP present in the test sample, a centrifugation step is used to remove bacteria from the sample. This may alter the bacterial cell wall, further releasing ATP and artificially increasing the measurement sensitivity of some bacteria to the screening compounds. Therefore, the proposed method is not only too time-consuming, but also insufficiently sensitive and reliable.

[0005] WO2019 / 162301 discloses a pharmaceutical composition comprising a first and a second peptide. The first peptide is a peptide of the bacteriocin PLNC8αβ. It also discloses a classical ATP efflux assay-based method for demonstrating PLNC8αβ-induced cell lysis. This method uses classical firefly luciferase, which is unstable at temperatures above 28°C. This instability impacts the reliability and sensitivity of the assay, as demonstrated by the lack of ATP release at peptide concentrations at or 2x the MIC.

[0006] The use of the classical luciferase FLE-50 in measuring ATP efflux has also been described by Lennart Nilsson ("New Rapid Bioassay of Gentamicin Based on Luciferase Assay of Extracellular ATP in Bacterial Cultures," Antimicrobial Agents and Chemotherapy, vol. 14, no. 6, 1 January 1978). As mentioned above, this instability prevents sensitive ATP measurements. Furthermore, the method described by Nilsson does not allow for real-time measurement of ATP efflux. The low sensitivity and signal amplitude necessitate a delay of at least 1 hour and 30 minutes before bioluminescence measurements can be performed, making this method suboptimal.

[0007] In view of the above, currently available methods for screening bactericides and testing bacterial susceptibility to known agents are time-consuming, insensitive and unreliable, and there is clearly a need for improved methods that are faster, more sensitive and more reliable. Summary of the Invention

[0008] In the context of the present invention, the inventors developed a method for detecting the efflux of ATP or its analogs from live bacteria in real time after exposure to novel candidate bactericidal compounds or known antibiotics using a luciferin-luciferase assay. It should be noted that the efflux of ATP or its analogs detected by the method of the present invention is not necessarily the result of whole-bacterial cell lysis or cell lysis accompanied by destruction of the bacterial cell wall (a phenomenon known for some antibiotics, such as β-lactams). As explained above, ATP is a fragile biomolecule that is rapidly hydrolyzed. This instability is important in culture media where ATP hydrolases may be present. Monitoring the reaction between luciferase and ATP in real time at a time point away from the bacterial cells avoids ATP degradation, maximizes detection sensitivity, and ensures measurement reliability. Furthermore, the adoption of real-time measurement eliminates the need for physical (e.g., high-speed centrifugation) or chemical (e.g., osmotic shock) stress on the bacteria. Because bacteria are simply grown to a minimum optical density at 600 nm (OD), luciferin-luciferase reagent and a candidate bactericidal compound or a known antibiotic are added, and bioluminescence is measured in real time (see FIG. 1), the results are not distorted by changes in the bacterial cell wall or changes in cell physiology due to medium changes (see FIG. 4, which would result in an artificial release of ATP). Thus, the adoption of real-time measurements significantly improves the sensitivity and reliability of the method according to the present invention compared to prior art methods.

[0009] Furthermore, by monitoring the reaction of luciferase with ATP or its analogs, which are also converted to light by luciferase, in real time, the inventors discovered, for the first time, the characteristic temporal characteristics and amplitude of ATP leakage for each antibiotic family (see Figure 8). Surprisingly, the inventors revealed that ATP leakage generally begins within minutes of contact with antibiotics at concentrations near the minimum inhibitory concentration (MIC) (see Table 2), and that ATP leakage is not necessarily the result of cell lysis, even while the bacteria are viable (see Figure 8). This discovery forms the basis for a method that is much faster than prior art methods, generally providing results after just a few minutes. Furthermore, the inventors have demonstrated that the method can be used with a wide range of antibiotics, either in purified form (see especially Figures 8, 10, 11) or as complex mixtures such as bacterial supernatants (see Figure 12), and with a wide range of bacterial strains (e.g., multidrug-resistant bacteria, see Figure 20) and morphologies (e.g., planktonic cells or biofilms, see Figures 8, 20).

[0010] Overall, the method designed by the inventors is thus very rapid (generally, a few minutes will be sufficient), sensitive (sub-MIC), reliable, and broadly applicable (for the compounds / compositions and bacterial species tested). Furthermore, the method can be performed in microplates and easily automated, making it industrially useful. It can also be implemented in microfluidic tools for ultra-high-throughput screening. Therefore, the method of the present invention is suitable for testing large libraries of novel candidate bactericidal compounds for drug discovery, as well as for rapid antibiotic susceptibility testing related to bacterial resistance.

[0011] Finally, we also demonstrated that by monitoring the reaction of luciferase with ATP or its analogs in real time, we were able to measure the time lag between the addition of antibiotics and the detection of ATP leakage, even when the antibiotic concentration was varied, enabling rapid and reliable evaluation of the minimum inhibitory concentration (MIC) of a compound for a given bacterial sample (see Figures 22 and 23).

[0012] According to a first aspect, the present invention provides a method for screening compounds for bactericidal activity, comprising the steps of: a) providing one or more test bacterial samples comprising live bacteria in a culture medium; b) adding a mixture of luciferin and thermostable luciferase to the test bacterial sample; c) adding a candidate composition to the test bacterial sample; d) incubating the test bacterial sample to which the mixture of luciferin and thermostable luciferase and the candidate composition have been added at 20 to 60°C, preferably 35 to 37°C, and measuring bioluminescence in real time; Including, the optical density at 600 nm (OD600) of the live bacteria in the test bacterial sample after steps a), b) and c) have been performed is at least 0.0002; wherein an increase in bioluminescence measured in the test bacterial sample in step d) indicates that the candidate composition added to the test bacterial sample in step c) contains at least one compound with bactericidal activity.

[0013] According to a second aspect, the present invention also provides a method for determining the susceptibility of a bacterial sample derived from a subject suffering from a bacterial infection to a group of known antibiotics, comprising the steps of: a) inoculating a culture medium with a bacterial sample from a subject suffering from a bacterial infection and, optionally, amplifying the bacteria in the sample; b) dividing the bacterial sample of step a) into a number of subsamples, the number of samples being at least equal to the number of known antibiotics to be tested; c) adding a mixture of luciferin and thermostable luciferase to each subsample; d) spiking one or more sub-samples with each of a group of known antibiotics or combinations of antibiotics; e) incubating a subsample containing a mixture of luciferin and thermostable luciferase and a known antibiotic at a temperature of 20 to 60°C, preferably 35 to 37°C, and measuring bioluminescence in real time; Including, the optical density at 600 nm (OD600) of the live bacteria in the subsample after steps a), b), c) and d) have been performed is at least 0.0002; wherein an increase in bioluminescence measured in the sub-sample in step e) indicates that the bacterial sample from the subject is sensitive to the added concentration of the known antibiotic added to the sub-sample in step d).

[0014] According to a third aspect, the present invention also provides a method for assessing the minimum inhibitory concentration (MIC) of a bactericidal compound, comprising the steps of: 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 subsamples; c) adding a mixture of luciferin and thermostable luciferase to said subsample; d) adding various concentrations of a bactericidal compound to said sub-samples; e) incubating the sub-sample to which the mixture of luciferin, thermostable luciferase and bactericidal compound has been added at 20-60°C, preferably 35-37°C, and measuring bioluminescence in real time, wherein the optical density at 600 nm (OD600) of the live bacteria in the sub-sample after steps a), b), c) and d) has been performed is at least 0.0002; f) for each concentration of bactericidal compound tested, determining the lag time between the time the bactericidal compound is added and the time an increase in bioluminescence signal is detected; g) expressing lag time as a function of bactericidal compound concentration; h) constructing an exponential decay curve fitted to the lag time measurement points as a function of bactericidal compound concentration; i) determining the lowest bactericidal compound concentration at the plateau of the exponential decay fitting curve; j) determining the lag time amplitude of the exponential decay fitting curve; k) assessing the MIC, wherein the MIC is: the antibiotic concentration corresponding to a lag time on the exponential decay fitting curve, or equal to (lag time at the plateau + 0.3 × lag time amplitude); The lowest bactericidal compound concentration at the plateau of the exponential decay fitting curve and and be evaluated as being included between A method comprising: [Brief explanation of the drawings]

[0015] [Figure 1] FIG. 1 shows a bioluminescence assay for real-time monitoring of ATP release. [Figure 2]Figure 1 shows a thermostable luciferase assay for real-time monitoring of ATP efflux. A: Stability test of the traditional luciferase assay. Bioluminescence signal obtained after preincubation of the reagent in rich broth (LB) or minimal (MOPS glucose 0.4%) medium at 28°C, followed by addition of ATP. B: Response of the classical bioluminescence assay to an antibiotic (neomycin) known to cause membrane damage at 28°C. Neomycin was added to logarithmic-phase culture medium of Escherichia coli (E. coli) MG1655 at its MIC, i.e., 22.8 μg / mL in LB and 0.8 μg / mL in MOPS glucose 0.4%. Controls without antibiotic in LB or MOPS glucose 0.4% are also included. C: Experiments performed with thermostable luciferase at 37°C. D: Thermostable luciferase is more suitable for ATP assays in minimal medium. Bioluminescence signals were obtained at various ATP concentrations using a thermostable luciferase in minimal medium or LB at 37°C. [Figure 3] This figure shows that the amount of live bacteria (reporter cells) in the assay is important. A: The assay was performed at 37°C with increasing amounts of E. coli reporter cells grown in Mueller-Hinton medium. The final OD600 values ​​of the cell culture media used are shown in each panel. B: The maximum recorded for the highest concentration of neomycin (100 μg / mL) is reported graphically. A final OD600 of 0.15 is a good fit for this assay. [Figure 4]A: This figure shows that centrifugation with a fresh medium change before aminoglycoside exposure enhances the drug's bactericidal effect. Staphylococcus aureus USA300 in TSB medium containing 0.25% glucose was incubated at 37°C until stationary phase was reached. Cells were either centrifuged at 12,000 rpm for 5 minutes (+) or placed at 37°C without centrifugation (≠). After centrifugation, the pellet was washed with fresh broth and placed at 37°C. Depending on the test, different concentrations of neomycin were added. S. aureus USA300 samples centrifuged and washed with fresh broth were more susceptible to neomycin. For example, bacterial CFUs were below the detection limit (102 CFU / mL) within 2 hours at 8 mM and 2 mM concentrations, but an additional 2 hours were required at 0.5 mM. Bar graphs represent the standard error of the mean (SEM) calculated using GraphPad Prism version 8.4.1. B: S. aureus USA300 in TSB medium containing 0.25% glucose was grown at 37°C until stationary phase was reached. Cells were centrifuged at 12,000 rpm for 5 minutes or placed at 37°C without centrifugation. After centrifugation, the pellets were washed with fresh broth and placed at 37°C. 100 μL of fresh medium containing 4 mM neomycin (final concentration: 2 mM) was added to all tubes, with or without centrifugation, at 5, 15, 30, 60, and 120 minutes after centrifugation. Samples were incubated with neomycin for a total of 2 hours, then washed three times with 1x PBS, diluted, and plated on tryptic soy agar. The following day, colony-forming units / mL were counted. Bar graphs represent standard deviations (SD) calculated using GraphPad Prism version 8.4.3. [Figure 5]This figure shows that the tested antibiotics alone generally do not affect the bioluminescence assay (thermostable luciferase). The luciferase-luciferin reagent was preincubated in the presence of various antibiotics in LB for 40 minutes at 37°C (in the absence of bacteria) before adding 30 nM ATP. A: RLU (maximum) at the highest concentration of a set of antibiotics used in this study. B: Evaluation of the effect of puromycin in the bioluminescence assay. At concentrations above the MIC (377 μg / mL), bioluminescence inhibition was strong, exceeding 50%. [Figure 6] This figure shows that the bioluminescence reagent maintained cell viability. The bioluminescence measurement reagent (thermostable luciferase) only slightly delayed the growth rate of MG1655 E. coli cells in LB. Cells were incubated on a microplate reader at 37°C in the presence or absence of the bioluminescence assay reagent, and cell growth was monitored by measuring the optical density. Measurements were performed in duplicate to ensure they were identical. The assay was performed under non-sterile conditions. [Figure 7] This figure shows the absence of ATP leakage by the aminoglycoside-resistant strain E. coli MG1655. The resistant strain (MG KanR) contains an expression gene for aminoglycoside O-phosphotransferase APH(3')-IIa. The figure shows the amount of ATP released when resistant (KanR) or non-resistant (WT) MG1655 E. coli strains were exposed to increasing concentrations of neomycin in MOPS minimal medium (0.4% glucose) at 37°C. No ATP leakage was observed in the resistant strain (KanR). [Figure 8]This figure shows that bactericidal antibiotics induced monophasic or multiphasic traces of ATP release. Representative traces of bioluminescence measurements obtained at 37°C after the addition of various antibiotics using E. coli reporter cells in LB growth medium. Bioluminescence signals were acquired for 1 second every 80 seconds. MIC values ​​are shown. Upper right panel: For neomycin, the killing curves obtained for different drug concentrations showed that cells remained viable for the first 90 minutes. Thus, with neomycin, biphasic ATP efflux was derived from viable cells. Biphasic ATP efflux was also observed for other aminoglycosides (kanamycin, streptomycin, amikacin, apramycin, and gentamicin). For neomycin, the signal at or near the MIC was lower than the signal at slightly lower concentrations. [Figure 9] Figure 1 shows that bactericidal antibiotics did not induce ATP release when used at non-bactericidal concentrations. Representative traces of bioluminescence measurements obtained at 37°C after antibiotic addition using E. coli reporter cells in LB growth medium. MIC values ​​are shown. Some bacteriostatic agents showed a strong signal only when used at high doses, which then became lethal. [Figure 10] A: Composite of the results shown in Figures 8 and 9. ATP leakage is a key signature of bactericidal antibiotic attack. A universal signal (dark gray) was detected when bactericidal antibiotics reacted with susceptible MG1655 E. coli. This signal was absent or very weak (light gray) with bacteriostatic antibiotics. B: A heatmap shows a composite of the analysis results for bactericidal and bacteriostatic antibiotics. The maximum amplitude of the bioluminescence signal was collected during the first 4 hours with drugs at (MIC) in LB and MOPS-G, or above (>MIC) in LB. Signal intensity is indicated by color intensity on a scale from strong (dark gray) to absent or very weak (light gray). Analysis was performed on GraphPad Prism 8.0.1. [Figure 11]Bioluminescence assay in minimal growth medium at 37°C. A: ATP release of E. coli grown in MOPS minimal medium (0.4% glucose) in the presence of antibiotics at their respective MICs in a set of bactericidal (left) and bacteriostatic (right). B: Full set of traces of gentamicin, neomycin, and apramycin (bacteriocidal), and rifampicin and azithromycin (bacteriostatic). Azithromycin stimulated ATP leakage at its highest concentration (400 μg / mL), consistent with that observed in rich medium (Figure 9). [Figure 12] This figure shows that the presence of neomycin in the supernatant of Streptomyces fradiae can be detected by the Gram-positive reporter strain M. luteus. ATP release from M. luteus grown in LB medium at 37°C in the presence of supernatant from a 4-day culture of S. fradiae. No ATP leakage was observed in a control experiment in the absence of the reporter M. luteus. [Figure 13] False-positive signals generated in rich medium. Black: Reporter bacteria E. coli (7 days) incubated at 37°C with luciferin-luciferase reagent solution and supernatant from a Streptomyces fradiae culture. Gray: Supernatant (no bacteria) incubated with fresh medium (to maintain constant volume) and luciferin-luciferase reagent solution alone. Light gray: Reporter bacteria E. coli incubated with fresh medium (to maintain constant volume) and luciferin-luciferase reagent solution alone (no supernatant). Bioluminescence was monitored for 210 minutes. [Figure 14] This figure shows that fresh LB medium induced bioluminescence when contacted with Streptomyces fradiae culture supernatant and luciferase-luciferin reagent. Addition of LB medium to supernatant from a 7-day Streptomyces fradiae culture resulted in a false-positive signal at 37°C. LB medium was added 30 minutes later (black arrow). Light gray: ATCC strains. Black: DSM strains (multiple mutant strain DSM41550 unable to produce neomycin). [Figure 15] This figure shows that the use of minimal medium overcomes the problem of false-positive signals generated in rich media. Supernatant from the neomycin-producing strain Streptomyces fradiae (WT, left side of the figure) was added, and ATP leakage was monitored in real time in MOPS-G medium at 37°C. Under these conditions, no bioluminescence signal was generated when reporter cells (E. coli) or WT Streptomyces fradiae cell supernatants were used alone. A signal was detected only when reporter cells were contacted with the supernatant from the drug-producing strain. A very weak signal was observed in the case of the Streptomyces fradiae mutant DSM (mutant DSM41550, right side of the figure), a multigene mutant unable to produce neomycin, suggesting the presence of some bactericidal compound other than neomycin. [Figure 16] Regarding assay optimization, A: shows the effect of incubating the supernatant with PMSF (0.1 mM concentration). 0.2 μL of PMSF dissolved in absolute ethanol was added to 19.8 μL of Streptomyces fradiae supernatant. The mixture was incubated at room temperature for 20 minutes. After incubation, the tubes were placed on ice until the microtiter plate was filled. Bioluminescence was monitored for 8 hours at 37°C. B: shows the effect of ultrafiltration using a membrane (cutoff 5000 Da). The supernatant was filtered using an ultrafiltration device (VivaSpin™ 500 GE HealthCare). Before filtration, the membrane was rinsed with buffer (MOPS-glucose 0.4%). The supernatant was placed in the concentration chamber. The concentration chamber and collector tube were centrifuged at 15,000 g for 20 minutes. The filtrate was collected and stored at -20°C. [Figure 17]Figure 1 shows a bioluminescence assay in minimal medium at 37°C for ATP release by E. coli cells grown in MOPS minimal medium (0.4% glucose) in the presence of increasing concentrations of neomycin. ATP leakage was slightly delayed compared to the neomycin trace obtained in Figure 8. The characteristic biphasic kinetics observed here, not seen in MOPS-glucose minimal medium, is due to the serial dilution of antibiotics performed in TSB medium to match the conditions used with the supernatant. [Figure 18] Bioluminescence assay in 384-well microtiter plates: ATP release from E. coli grown in MOPS minimal medium (0.4% glucose) in the presence of increasing concentrations of neomycin. The assay was performed in reduced volume in white 384-well microtiter plates at 37°C. [Figure 19] Figure 16. Bioluminescence assay for detecting the presence of bactericidal compounds in culture supernatants of drug-producing strains. A: Principle of the method applied to the supernatant of a drug-producing strain. B: Optimization of the assay with filtration of the supernatant as shown in Figure 16b. Bioluminescence was monitored for 4 hours at 37°C. C: Test using duplicate culture supernatants of Streptomyces fradiae using the optimized protocol. Bioluminescence signals were detected for the neomycin-producing wild-type strain ATCC of Streptomyces fradiae but not for the non-producing multi-mutant strain (DSM41550) or the single-mutant strain (Δneo6). [Figure 20]Figure 1 shows drug-dependent ATP release from biofilms of multidrug-resistant Staphylococcus aureus USA300. A: Control experiment at 37°C in the absence of antibiotic. Biofilms were washed with MOPS medium before exposure to neomycin in a bioluminescence assay. Biofilms were obtained as described in the Examples section and washed twice with MOPS-G medium. The wash solutions were tested for the presence of ATP in a bioluminescence assay. These solutions did not contain ATP. The washed biofilms were then tested for ATP release. A signal was present during the first 40 minutes. This ATP release likely results from the physical disruption of the biofilms while shaking them in the plate reader. B: Response of biofilms to drug exposure at 37°C. [Figure 21] FIG. 1 shows a bioluminescence assay for antibacterial agent discovery. [Figure 22] Figure 2 shows the analysis of the variation of lag time as a function of drug concentration and positioning of MIC values. The value of lag as a function of antibiotic concentration (c) is analyzed as an exponential decay Y = (YO - plateau) * exp(-K * c) + plateau. The experimental values ​​of MIC measurements are displayed on the curve by open circles. For each antibiotic, a value X was calculated and these values ​​are displayed in Table 2. [Figure 23] Representative analysis of lag time as a function of increasing antibiotic concentrations. Experiments were performed using E. coli strains grown in rich LB medium or, where indicated, cation-adjusted Mueller-Hinton (MH) medium. The area of ​​appearance of the bioluminescence signal for amikacin (Figure 8) is shown as an example (left). For each drug concentration, the time of signal increase is indicated by an arrow. The corresponding fit of the data is shown on the right. Data were analyzed using the equation in Figure 22. For each antibiotic, the experimentally determined MIC value is displayed as an open circle. [Figure 24]Figure 1 shows a comparative assay of conventional (non-thermostable) firefly luciferase and Kikkoman's thermostable luciferase. Both enzymes were incubated at 37°C in rich medium (LB) or minimal medium (MOPS-glucose), and luciferase activity was measured at various times. Error bars represent the standard error of the mean of three independent assays. [Figure 25] Bioluminescence signal obtained with E. coli in clinical Muller growth medium. A: Hinton medium after addition of neomycin. Following EUCAST recommendations, an inoculum of 0.000275 bacterial colony-forming units per mL (approximately 5.5 x 10 CFU / mL) was used at D600. B: The curve was smoothed using the moving average method. MIC estimation. The experimental MIC (3.12 μg / mL) is indicated by an open circle, and the X value at the MIC is shown along with the standard error of the mean for four independent experiments. The bars represent the standard deviation (SD) calculated for four independent experiments. DETAILED DESCRIPTION OF THE INVENTION

[0016] As used herein, the terms "comprising" (and any form of "comprising," such as "comprise" and "comprises"), "having" (and any form of "having," such as "have" and "has"), "including" (and any form of "including," such as "includes" and "include"), or "containing" (and any form of "containing," such as "contains," "contain") are open-ended and mean not excluding additional, unrecited elements or method steps. The phrase "consisting essentially of" means excluding other ingredients or steps of an essential meaning. Thus, a composition consisting essentially of the recited ingredients would not exclude minor ingredients, contaminants, and pharmaceutically acceptable carriers. "Consisting of" shall mean excluding more than trace elements of other ingredients or steps. Whenever the term "comprising" (or any of its derivatives such as "comprise" and "comprises") is used in this specification, the present invention also relates to the same embodiments in which "comprising" (or any of its derivatives such as "comprise" and "comprises") is replaced with "consisting essentially of" or "consisting of."

[0017] Methods for screening compounds for bactericidal activity (screening methods) As described above, we have developed a method for detecting the real-time efflux of ATP or its analogs from live bacteria after exposure to novel candidate bactericidal compounds or known antibiotics using a luciferin-luciferase assay. Our method is very rapid (a few minutes will generally be sufficient), sensitive and reliable (due to the real-time measurement, which prevents distortion due to fragile ATP degradation and eliminates physical and chemical stress on bacterial cells), and broadly applicable (in terms of the types of compositions and bacteria tested). Furthermore, it can be easily automated by implementing it in microplates, making it industrially useful, or it can be implemented in microfluidic tools for ultra-high-throughput screening. Thus, our method allows for the screening of large libraries of novel candidate bactericidal compounds.

[0018] According to a first aspect, the present invention provides a method for screening compounds for bactericidal activity, comprising the steps of: a) providing one or more test bacterial samples comprising live bacteria in a culture medium; b) adding a mixture of luciferin and thermostable luciferase to the test bacterial sample; c) adding the candidate composition to the test bacterial sample; d) incubating the test bacterial sample to which the mixture of luciferin and thermostable luciferase and the candidate composition have been added at 20 to 60°C, preferably 35 to 37°C, and measuring bioluminescence in real time; Including, the optical density at 600 nm (OD600) of the live bacteria in the test bacterial sample after steps a), b) and c) have been performed is at least 0.0002; wherein an increase in bioluminescence measured in the test bacterial sample in step d) indicates that the candidate composition added to the test bacterial sample in step c) contains at least one compound with bactericidal activity.

[0019] Step a): Providing at least one test bacterial sample containing live bacteria in a culture medium Optical density of test bacterial samples The inventors have demonstrated that the amplitude of the ATP leakage signal correlates with the amount of viable bacteria (also referred to herein as "reporter cells") in the culture medium of the final sample (the test bacterial sample after steps a), b), and c) have been performed. To detect ATP leakage, bacteria must be used whose optical density at 600 nm (OD600) in the final sample is at least 0.0002, preferably at least 0.0003, at least 0.0005, at least 0.001, at least 0.005, at least 0.01, more preferably at least 0.015, and even more preferably at least 0.1. Initially, the inventors used black microplates and found that a final OD600 of 0.3 or 0.15 provided acceptable ATP leakage signals, but a final OD600 of 0.015 provided weak ATP leakage signals, making results unreliable. Furthermore, a final OD600 of 0.0015 provided too weak an ATP leakage signal to be useful. Using a supplementary assay to improve the sensitivity of the screening method using white microplates (Greiner Bio-one, 655075), the inventors found that the ATP leakage signal could be acceptable even when the bacterial concentration in the final sample was very low. Thus, the inventors found that the ATP leakage signal was detectable at an OD of at least 0.0002, preferably at least 0.0003 (Figure 25). Thus, after steps a), b) and c) have been performed, the OD600 of the live bacteria in the test bacterial sample is at least 0.0002, preferably at least 0.0003, at least 0.0005, at least 0.001, at least 0.005, at least 0.01, more preferably at least 0.015, preferably 0.0002-0.5, 0.0003-0.5, 0.0005-0.5, 0.001-0.5, 0.005-0.5, 0.01-0.5, also 0.015-0.5, 0.03-0.5, 0.05-0.5, or 0.1-0.5.Furthermore, since the results obtained when the final OD600 is 0.3 or 0.15 are particularly satisfactory, according to a preferred embodiment of the screening method of the present invention, the OD600 of the live bacteria in the final sample is comprised between 0.1 and 0.3, or between 0.1 and 0.2. Preferably, the OD600 of the live bacteria in the final sample is 0.15.

[0020] Therefore, the initial OD600 of live bacteria in the test bacterial sample in step a) (before performing steps b) and c)) is higher than 0.03, preferably higher than 0.05, higher than 0.1, more preferably higher than 0.3. Preferably, the volumes of the mixture of luciferin and thermostable luciferase added in step b) and the candidate composition added in step c) (after steps b) and c)) are such that the final volume is 1.5 to 3 times, for example 2 times, the initial volume of the test bacterial sample. In this case, the initial OD600 of the live bacteria in the test bacterial sample in step a) is therefore 1.5 to 3 times (e.g., 2 times) higher than 0.0002, preferably 0.0003, 0.0005, 0.001, 0.005, 0.01, or 0.015, more preferably 1.5 to 3 times (e.g., 2 times) higher than 0.05, 1.5 to 3 times (e.g., 2 times) higher than 0.1, and even more preferably 1.5 to 3 times higher than 0.15. Those skilled in the art will be able to easily select the initial OD600 of the initial test bacterial sample in step a) depending on the required amounts added in steps b) and c) and the target final OD600 of the live bacteria in the final sample cultured in step d). When the additions in steps b) and c) result in a final volume that is twice the initial volume of the test bacterial sample in step a), the initial OD of the live bacteria in the test bacterial sample in step a) is preferably at least 0.0004, at least 0.0006, at least 0.001, at least 0.002, at least 0.01, at least 0.02, at least 0.03, at least 0.1, more preferably 0.2 to 0.6, or 0.2 to 0.4, and most preferably about 0.3.

[0021] In the context of the present invention, the term "optical density at 600 nm (OD600)" refers to the measurement of bacterial growth by optical density at 600 nm. This measurement is based on the absorbance detection mode, which basically measures the fraction of light that passes through a bacterial sample. Particles in a solution scatter light, and the more particles (bacteria) there are in a solution, the more light is scattered by the particles. Therefore, as the bacterial population replicates, the scattering of light increases, and the absorbance reading increases. At the same time, this means that the absorbance mode is only utilized to measure the degree of light scattering, instead of measuring the physical absorption of light energy by absorbing molecules. OD 600 is thus a measure of light scattering, and OD 600 The value can be directly correlated to the number of microorganisms.

[0022] Preferably free from mechanical and chemical stress Furthermore, it is important to emphasize that the screening methods of the present invention preferably avoid manipulation of live bacteria. In particular, the methods preferably exclude chemical or mechanical manipulations that may disrupt or damage the physiology of live bacteria, i.e., manipulations that may induce chemical or mechanical stress on the live bacteria in the sample. Mechanical and chemical stress, like bactericidal compounds, damage bacterial structures, particularly bacterial membranes and their function. Therefore, analyzing bacterial cells that have previously been subjected to chemical and / or mechanical stress can artificially increase the bacterial sensitivity to the antibiotic compounds measured, distorting the results (see Figure 4).

[0023] Mechanical stress can be defined as stress caused by physically manipulating live bacteria in a bacterial sample, for example, during transport, storage (e.g., on ice), or centrifugation of the sample. Some strains, such as Pseudomonas aeruginosa, have been shown to be sensitive to centrifugation (Gilbert et al., 1991, Centrifugation injury of Gram-negative bacteria). Therefore, including a step of subjecting bacteria used in a screening method for bactericidal compounds to mechanical stress can lead to unreliable results.

[0024] Chemical stress refers to the addition of a reagent to a bacterial culture medium at a concentration that alters the physiological balance between live bacteria and their environment, resulting in a sudden change in the solute concentration around the bacteria and a sudden change in the movement of water across the cell membrane (i.e., osmotic shock). For example, as Raulin de la Roy et al. (J. Biolumin. Chemilumin. 6, 193-201 (1991)) found, the strong dilution of bacteria contained in the culture medium in distilled water results in an osmotic shock, which alters the bacterial structure, particularly the bacterial membrane, and its function, potentially artificially increasing the bacterial sensitivity to antibiotic compounds.

[0025] Therefore, according to one embodiment of the screening method of the present invention, the live bacteria in the test sample provided in step a) have not been subjected to mechanical or chemical stress prior to step a), in particular for less than 2 hours, preferably less than 4 hours, more preferably less than 6 hours. Specifically, the live bacteria in the test sample provided in step a) have preferably not been subjected to centrifugation or osmotic shock. More specifically, the live bacteria in the test sample provided in step a) have not been subjected to centrifugation or osmotic shock prior to step a), in particular for less than 2 hours, preferably less than 4 hours, more preferably less than 6 hours. According to a more preferred embodiment, the bacterial sample is not subjected to mechanical or chemical stress during the screening method of the present invention.

[0026] The types of bacteria present in the bacterial test sample As indicated above, an advantage of the screening method of the present invention is that it allows for the screening of bactericidal compounds against a wide range of bacterial strains and growth conditions. According to one embodiment, the live bacteria in the test sample are selected from the group consisting of antibiotic-resistant bacteria, including multidrug-resistant bacteria, pathogenic bacteria, planktonic cells, and bacterial cells in biofilms. In the context of the present invention, the expression "antibiotic-resistant bacteria" refers to bacterial strains that have developed the ability to overcome the bactericidal and / or bacteriostatic effects of antibiotics, such that the bacteria are not killed and continue to grow in the presence of bactericidal and / or bacteriostatic compounds. Antibiotic-resistant bacteria can be resistant to more than one antibiotic, and bacteria that are resistant to multiple antibiotics are called "multidrug-resistant bacteria." In the context of the present invention, the term "pathogenic bacteria" refers to all bacterial strains that are capable of causing damage and / or pathology to living organisms, in particular animal, more particularly mammalian, and even more particularly human subjects. Furthermore, as used herein, the term "planktonic cells" refers to bacteria that are free-flowing in suspension. Planktonic cells are in contrast to "biofilm cells," which can be defined as a structured community of bacterial cells encased in a self-produced polymeric matrix and attached to an inert or living surface.

[0027] Some bacterial genera and, in particular, some bacterial strains are of particular medical interest and can therefore be suitably used in the screening method according to the present invention. These include, for example, the ESKAPE group of antibiotic-resistant "priority pathogens" defined by the World Health Organization (WHO). ESKAPE is an acronym for the designation of the following Gram-positive and Gram-negative species: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species. The WHO has also published a catalog of 12 bacterial families that pose the greatest threat to human health. These are: Helicobacter pylori (clarithromycin-resistant), Campylobacter spp. (fluoroquinolone-resistant), Salmonellae (fluoroquinolone-resistant), Neisseria gonorrhoeae (cephalosporin-resistant, fluoroquinolone-resistant), Streptococcus pneumoniae (penicillin-nonsusceptible), Haemophilus influenzae (ampicillin-resistant), Shigella spp. (fluoroquinolone-resistant). Also of interest are, for example, coagulase-negative staphylococci, Mycobacterium tuberculosis, many Streptococcus species (e.g., Streptococcus pneumoniae, Streptococcus pyogenes) and Pseudomonas species (e.g., Pseudomonas aeruginosa), Enterococcus faecalis, Escherichia coli, Proteus mirabilis, Serratia marcescens, and Citrobacter freundii.

[0028] Other bacterial genera / strains are particularly easy to manipulate (e.g., because they are non-pathogenic and do not require high-security biocontainment) and may be preferably used in the screening methods of the present invention (once a new bactericidal compound is identified using such bacteria, its bactericidal effect on other bacteria, pathogenic or not, can be confirmed. These include non-pathogenic strains of E. coli, Streptococcus, Bacillus subtilis, and attenuated Salmonella strains. Some bacteria are particularly sensitive to many antibiotics and can be particularly useful in antibacterial screening, such as the Gram-positive strain Micrococcus luteus.

[0029] Culture medium As noted above, in the test sample, live bacteria are present in the culture medium. The culture medium should be capable of maintaining the viability of the bacterial cells over the measurement period, but many different culture media can be used and one skilled in the art will understand which medium to choose depending on the type of bacterial reporter cells used in the screening method. In particular, when screening purified chemicals (described below), both rich media (e.g., Luria Broth (LB), cation-adjusted Mueller-Hinton (MH)) and minimal media supplemented with a carbon source such as glucose (e.g., MOPS-glucose) can be used.

[0030] However, when screening complex mixtures such as bacterial supernatants, a minimal medium supplemented with a carbon source such as glucose (e.g., MOPS-glucose) would preferably be used, since the inventors surprisingly discovered that the combination of a rich medium (e.g., Luria Broth (LB)) with bacterial supernatants induced the generation of bioluminescence from luciferin by luciferase, even in the absence of bacterial reporter cells (see Figures 13 and 14), whereas this effect was not observed when a minimal medium supplemented with glucose (e.g., MOPS-glucose, see Figure 15) was used.

[0031] In the context of the present invention, "minimal medium" refers to a medium containing the minimum necessary for the growth of the target bacterium. Generally, it contains only inorganic salts, a carbon source, and water. Examples of minimal media that can be used in the present invention when supplemented with a carbon source such as glucose include MOPS and M9. MOPS medium contains a mixture of potassium morpholinopropanoic acid sulfonate, potassium hydrophosphate, water, and thiamine, preferably adjusted to approximately pH 7, to which other components (ferrous sulfate, ammonium chloride, potassium sulfate, calcium chloride, magnesium chloride, and sodium chloride) are added. Methods for preparing the medium will be understood by those skilled in the art. The minimal medium (MOPS) described herein is commercially available from Teknova, Inc., 2290 Bert Drive, Hollister, CA 95023 USA. Teknova also offers the same minimal medium in a concentrated form to reproduce high growth rates similar to those obtained with rich media such as LB. When a minimal medium is used, it is preferable to supplement it with a carbon source such as glucose, and the medium is used at a concentration containing 0.4 to 2% glucose.

[0032] In the present invention, the term "rich medium" refers to a medium that allows bacteria to grow at the maximum rate. In addition to the components of minimal medium, it generally contains additional components, specifically sodium chloride, complex nutrients (such as a peptide mixture (tryptone) and yeast extract). Examples of rich media that can be used in the present invention include Luria Broth (LB) (also known as Lysogeny Broth (LB)) and cation-adjusted Mueller-Hinton (MH) medium. LB medium is a nutrient-rich medium primarily used for bacterial growth. Its composition may vary slightly depending on the provider, but it always contains the following: peptide and casein peptone, vitamins (including vitamin B), trace elements (such as nitrogen, sulfur, and magnesium), and minerals. According to a preferred embodiment, cation-adjusted Mueller-Hinton (MH) medium is used for its high reproducibility. It contains adjusted levels of magnesium and calcium, which are important components of bacterial membranes. It is commercially available from Merck and contains beef extract, casein, and acid hydrolysate of starch.

[0033] Step b): Addition of a mixture of luciferin and thermostable luciferase to the above sample "Luciferase" is a general term for an oxidase that produces bioluminescence. Luciferase does not require an external light source but does require the addition of luciferin as a substrate. Luciferase promotes a chemical reaction that combines molecular oxygen with luciferin to form oxyluciferin, simultaneously emitting a photon. This reaction requires energy provided by adenosine triphosphate (ATP) or certain analogs thereof (such as diadenosine tetraphosphate (Ap4A), which reacts weakly with luciferase and dehydroluciferin) (Garrido et al., J. Biochem. Biophys. Methods 30 (1995) 191-198), resulting in the emission of light (bioluminescence). Bioluminescence is proportional to the concentration of ATP and ATP analogs hydrolyzable by luciferase, and therefore can be measured. Therefore, luciferase can be used to detect cellular ATP and specific ATP analog levels in cell viability assays or kinase activity assays. Classically used firefly luciferases use luciferin as a substrate, which is oxidized to oxyluciferin in a reaction utilizing molecular oxygen and ATP (or certain ATP analogs, as described above), emitting light at 560 nm. Magnesium ions may also be required for this reaction to occur (depending on the specific luciferase / luciferin pair used). Because oxygen and magnesium ions are already present in the bacterial sample added in step a), only luciferase and luciferin need be added in step b) to measure leakage of ATP and ATP analogs by the bactericidal compound.

[0034] In the context of the present invention, we first evaluated the ability of classically used firefly luciferase (Invitrogen Molecular Probes, referenced as A22066) to detect ATP or its analogs in rich and minimal media. Addition of ATP or its analogs to rich (Luria Broth (LB)) or minimal (MOPS supplemented with glucose) media containing the luciferin / luciferase reagent induced luminescence. Initially, we determined that the reagent was relatively stable in rich LB media and concluded that firefly luciferase could potentially be used to detect ATP signals. This assay was performed at 28°C. However, at the same temperature, in minimal MOPS media supplemented with glucose, the reagent was rapidly inactivated, and the resulting ATP signal was unstable and difficult to detect. To determine whether classically used firefly luciferases allow for sensitive and reliable measurement of ATP at temperatures typically used for bacterial growth, the inventors performed complementary assays in rich and minimal media at 37°C. The results, shown in Figure 24, indicate that 37°C is not optimal for obtaining reliable and sensitive ATP detection in minimal and rich media using conventional firefly luciferases. The inventors surprisingly found that this problem does not occur with thermostable luciferases. Therefore, the screening method of the present invention uses a thermostable luciferase that is added to the sample prepared in step a) of the method.

[0035] In the context of the present invention, the term "thermostable luciferase" relates to a luciferase enzyme, particularly a firefly luciferase, that has improved stability at temperatures above 30°C. In particular, the thermostable luciferase preferably has improved activity at temperatures above 30°C, particularly at 37°C, compared to conventional luciferases, particularly compared to the wild-type firefly luciferase from Photinus pyralis, published in GenBank release 243 under accession number AAA29795.1. Improved thermostability of luciferases can be obtained by inserting various mutations into the amino acid sequence of the luciferase or by combining the sequences of various luciferases to create chimeras. For example, Kikkoman's thermostable luciferase was created by genetic chimerization of Photinus pyralis luciferase with a thermostable mutant of Luciola cruciata luciferase, and by combining mutations to improve catalytic performance (Hirokawa et al., Biochimica et Biophysica Acta 1597 (2002) 271-279). Several types of mutant luciferases with improved stability have been disclosed (see, for example, EP0524448, US5229285A, and US6074859 (all Kikkoman Corporation), and NanoLuc (registered trademark) (NLuc) manufactured by Promega (Hall et al., ACS Chem Biol. 2012 Nov 16;7(11):1848-57. doi:10.1021 / cb3002478, England et al., Bioconjug Chem. 2016 May 18;27(5):1175-1187. doi:10.1021 / acs.bioconjchem.6b00112)), and can be used in the screening method of the present invention.

[0036] According to one embodiment of the screening method of the present invention, the thermostable luciferase is selected from the group consisting of thermostable firefly luciferases manufactured by Kikkoman Corporation (e.g., those disclosed in EP052448A1 or US5229285, including the thermostable firefly luciferase in the CheckLite AT100 kit), or luciferase Nanoluc (also referred to as "NLuc") manufactured by Promega (see Hall MP, Unch J, Binkowski BF, et al. Engineered luciferase reporter from a deep-sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chem Biol. 2012;7(11):1848-1857. doi:10.1021 / cb3002478; the sequence of Nanoluc is disclosed in Figure S6 and has the accession number in GenBank release 243AF179290), or other luciferases that are thermostable in the medium used for screening. In particular, the activity of luciferase in the medium used for screening is tested. Preferably, the thermostable luciferase is thermostable firefly luciferase from Kikkoman Corporation (e.g., as disclosed in EP0524448A1, US522985A or US6074859, which is obtained by adding T219I and V239I substitutions to the thermostable firefly luciferase from CheckLite AT100 kit (having the accession number of GenBank release 243 AAE43251.1), which corresponds to SEQ ID NO: 7 of US6074859), or modified luciferase NanoLuc® from Promega.

[0037] In the screening method of the present invention, the thermostability of luciferase advantageously allows testing in several types of media, prevents luciferase degradation when monitoring long-term reactions, and facilitates handling of the enzyme during preparation. As used herein, "luciferin" is a general term for compounds present in living organisms that produce bioluminescence and emit light after oxidation by luciferase. Luciferin typically undergoes enzyme-catalyzed oxidation, and the resulting excited-state intermediate decays to the ground state, resulting in light emission. Thus, luciferin is a low-molecular-weight compound that serves as a substrate for luciferase enzymes. There are various types of luciferin (D-luciferin and its derivatives, such as CycLuc1 and AkaLumine-HCl, coelentarazin and its derivatives, such as diphenylterazine and selenoterazine, and furimazine) and its derivatives / analogs (e.g., D-luciferin derivatives / analogs: CycLuc1 and AkaLumine-HCl, coelentarazin derivatives / analogs: diphenylterazine and selenoterazine). Luciferin derivatives / analogs can also be used. In particular, luciferin analogs have been generated that react with luciferase mutants to emit light at wavelengths other than 560 nm, such as 599, 607, 675, and 706 nm (Jathoul et al., Angew Chem Int Ed Engl. 2014 Nov 24;53(48):13059-63. doi: 10.1002 / anie.201405955). The analog AkaLumine-HCl produces bioluminescence in the near-infrared wavelength range (677 nm) (Kuchimaru et al., Nat Commun. 2016 Jun 14;7:11856. doi: 10.1038 / ncomms11856). Other novel luciferins, when combined with engineered luciferases, also produce light at 460 nm (Hall et al., ACS Chem Biol. 2012 Nov 16;7(11):1848-57. doi: 10.1021 / cb3002478) or 677 nm (Yeh et al., Nat Methods. 2017 Oct;14(10):971-974. doi: 10.1038 / nmeth.4400). Thus, various types of luciferin / luciferase reactions can be used, but these reactions always require the presence of molecular oxygen and ATP or its analogs.Examples of luciferins that can be used in the present invention include luciferin analogs in any luciferin-luciferase pair, provided that the luciferase is thermostable. Examples of pairs such as furimazine-Nanoluc are listed in Table 1 of Yeh et al., Nat Methods. 2017 Oct;14(10):971-974. doi: 10.1038 / nmeth.4400.

[0038] The combination of luciferin and thermostable luciferase used in the screening method of the present invention is selected so that the selected luciferin is an appropriate or optimal substrate for the selected thermostable luciferase. Examples of appropriate (luciferin / thermostable luciferase) combinations include (firefly luciferin / thermostable firefly luciferase), furimazine-Nluc luciferase, furimazine-Antares luciferase, diphenylterazine (DTZ)-teLuc, diphenylterazine (DTZ)-Antares2, and coelenterazine (STZ)-yeLuc.

[0039] Kits containing a solution containing luciferin and thermostable luciferase in an appropriate ratio are available in the art, examples of which include the CheckLite AT100 kit available from Kikkoman and Nanoluc from Promega.

[0040] The concentrations of luciferin and luciferase added in step b) or the corresponding dilution factors of the stock luciferin and luciferase solutions will be adjusted by the skilled artisan so that the final concentration after the candidate composition is further added in step c) corresponds either to the recommended final luciferin and luciferase concentrations described in the instructions accompanying the selected commercially available luciferin / luciferase kit, or to an optimal concentration of luciferin / luciferase selected based on preliminary measurements using conventional methods known in the art for this purpose.

[0041] In a preferred embodiment of the screening method of the present invention (described in the Examples) using the thermostable luciferase kit "CheckLite AT100" (Kikkoman Corporation) or NanoLuc (registered trademark) (NLuc), the mixture of luciferin and luciferase after steps a) to c) have been performed (in the final sample containing all reagents) is desirably diluted 6 to 9 times compared to the initial stock solution prepared from the Kikkoman Corporation kit according to the art and manufacturer's guidelines.

[0042] Step c): Adding a candidate composition to the at least one sample After step b) of adding a luciferin / thermostable luciferase mixture to a sample containing live bacteria in culture medium, a candidate composition (containing at least one compound screened for bactericidal effect) is added in step c). The methods of the present invention allow for the screening of multiple candidate compositions that differ in structure and origin. According to one embodiment of the screening methods of the present invention, the candidate composition is a purified compound, which may be a chemical or biological compound. In the context of the present invention, a "purified compound" refers to a compound obtained by physically separating a chemical substance of interest from contaminants or pollutants. The purified compound screened by the method of the present invention can be obtained by any one of known purification methods. A purified compound represents at least 90% w / w, preferably at least 95% w / w, at least 96% w / w, at least 97% w / w, at least 98% w / w, or at least 99% w / w of the candidate composition.

[0043] As used herein, the term "compound" refers to a substance in which two or more different types of atoms (chemical elements) exist in a certain stoichiometric ratio. The chemical compounds screened by the method of the present invention may be derived from natural products, i.e., those that exist in nature as they are, or may be artificial compounds obtained by chemical synthesis. As used herein, the term "biological compound" refers to an organic compound, i.e., a compound containing carbon. Biological compounds may be obtained from living organisms or synthesized by chemical synthesis. In a preferred embodiment of the method of the present invention, the biological compound included in the candidate composition may be selected from DNA (any type of DNA, including single-stranded DNA, double-stranded DNA, hairpin DNA, linear or circular DNA, etc.), RNA (any type of RNA, including mRNA, miRNA, siRNA, single-stranded RNA, double-stranded RNA, hairpin RNA, etc.), proteins (enzymes, antibodies, toxins, etc.), peptides, hormones, metabolites, and microorganisms (including viruses, particularly phages). In a preferred embodiment of the method of the present invention, the biological compound included in the candidate composition is a phage. Here, the term "phage" or "bacteriophage" refers to a virus that infects and replicates within bacteria. According to another embodiment, the candidate composition added in step c) of the method of the invention is a complex mixture, preferably a bacterial supernatant or extract.

[0044] In the context of the present invention, the term "bacterial supernatant" refers to the liquid above the solid bacterial material sedimented by sedimentation, settling, or centrifugation of a bacterial culture. Thus, the bacterial supernatant includes products secreted by the cultured bacteria in addition to the culture medium. Furthermore, the term "bacterial extract" refers to a product obtained by extracting one or more bacterial components (including proteins, lipids, metabolites, and / or sugars) from a bacterial culture. Preferably, when a bacterial extract is used in the screening method of the present invention, ATP possibly present in the extract is hydrolyzed before screening begins. This inactivation can be performed in different ways known in the art. For example, ATP may be inactivated by heating the bacterial sample at a temperature between 37 and 50°C. Those skilled in the art will be able to determine the heating temperature to inactivate ATP without altering other compounds in the bacterial extract to be tested in the subsequent measurement. Another method for inactivating ATP present in a sample before adding the screened compound includes the use of enzymes capable of digesting ATP. Examples include ATPase (which converts ATP to ADP (adenosine diphosphate), which is then converted to AMP (adenosine monophosphate)) and adenosine monophosphate deaminase (which removes amino groups). Generally, after the enzyme digests ATP, these enzymes should be removed from the test sample so as not to digest the ATP released after the addition of the compound to be screened. To achieve this, a surfactant such as that used in the Kikkoman Corporation kit disclosed above can be used. Alternatively, these enzymes may be removed by filtration. Protein filtration can be performed using any conventional method. In particular, the ultrafiltration device (VivaSpin™ 500 GE HealthCare) used in the present invention can be used to filter the supernatant.

[0045] In an embodiment of the method of the present invention, the candidate composition is filtered prior to its addition in step c) using a membrane with a cutoff of preferably up to 15,000 Da, preferably 2,000-15,000 Da, 3,000-10,000 Da, 4,000-8,000 Da, e.g., 5,000 Da. Such a filtration step is particularly useful when the candidate composition is a complex mixture, such as a bacterial supernatant or bacterial extract. Indeed, the inventors have shown that large molecules present in bacterial supernatants interfere with the reaction by which luciferase converts ATP or its analogs to bioluminescence, and that prior filtration of the bacterial supernatant through a membrane with a 5,000 Da cutoff restores optimal conversion of ATP or its analogs to bioluminescence by luciferase (see Figure 19b). Thus, if the candidate composition is a complex composition such as a bacterial supernatant or bacterial extract, prior to its addition in step c) it is preferably filtered through a membrane having a cutoff of up to 15,000 daltons, preferably comprised between 2,000 and 15,000 daltons, 3,000 and 10,000 daltons, 4,000 and 8,000 daltons, for example 5,000 daltons. In any event, if the candidate composition includes at least one bactericidal compound, it is preferably added at a concentration expected to result in leakage of ATP.

[0046] Compounds that qualify as bactericidal generally have an MIC of at least 0.002 μg / mL and at most 1024 μg / mL. Accordingly, the candidate composition (especially when comprising a purified compound) will preferably be added in step c) at a concentration such that the final concentration in step d) (after addition of both the mixture of luciferin and thermostable luciferase and the candidate composition to the test bacterial sample) is at least 0.002 μg / mL, and preferably the concentration of the candidate composition at the start of step d) is between 0.002 μg / mL and 1024 μg / mL.

[0047] Preferably, after steps a), b) and c) have been performed, taking into account the preferred concentrations disclosed above for live bacteria, luciferin, luciferase and candidate composition, The OD600 of the live bacteria is at least 0.1, preferably between 0.1 and 0.3, more preferably between 0.1 and 0.2, in particular 0.15. The luciferin solution is diluted to the concentration recommended by the luciferin manufacturer or to the optimal concentration based on preliminary experiments (6-9 times if using Kikkoman's "CheckLite AT100" kit or NanoLuc® (NLuc)). The thermostable luciferin solution is diluted to the concentration recommended by the luciferin manufacturer or to the optimal concentration based on preliminary experiments (6-9 times when using Kikkoman's "CheckLite AT100" kit or NanoLuc® (NLuc)). And / or The concentration of the candidate composition is at least 0.002 μg / mL, preferably between 0.002 μg / mL and 1024 μg / mL.

[0048] Step d): Incubate the sample at 20-60 °C, preferably 35-37 °C, and measure bioluminescence in real time (measurement step). After steps b) and c), the sample is then incubated during step d) at a temperature of 20-60°C (a temperature selected consistent with the thermostability parameters of the luciferase), preferably 30-40°C, and more preferably 35-37°C. Such conditions are adequate for the survival of the sample's bacteria in the absence of a bactericidal compound. In this way, only ATP leakage due to the presence of a bactericidal compound in the candidate composition is detected. While these temperature conditions are believed to be optimal for the thermostable luciferases used in accordance with preferred embodiments of the screening method of the present invention, the temperature can be adapted to match the thermostability parameters of each luciferase.

[0049] During this step, bioluminescence is measured in real time, and a detected increase in measured bioluminescence indicates that the candidate composition added to the sample in step c) contains at least one compound with bactericidal activity.

[0050] Bioluminescence can be measured in real time by any means known to those skilled in the art for such measurements.In particular, bioluminescence can be measured by a means selected from the group of instruments called luminometers, such as the following examples: single-tube luminometers (Luminescencer Octa, ATTO, Lumat 3, Berthold Technologies, Sirius L, TiterTek Berthold, etc.), multi-tube luminometers (AutoLumat LB 953, Berthold Technologies), real-time culture luminometers (Kronos Dio, ATTO), high-throughput screening systems (FDSS7000EX, Hamamatsu), particularly microtiter plate readers (e.g., InfinitePro200, TECAN, FDSS / RayCatcher, Hamamatsu, Plate Chameleon V, Hidex, Synergy H1, BioTec), etc.

[0051] As used herein, "measuring in real time" means that multiple measurements of the same sample are performed during a predetermined period (measurement period) to provide a record of signal changes as a function of time with sufficient time resolution. This period can be 20 to 180 minutes, preferably 20 to 60 minutes, and more preferably 15 to 40 minutes. Depending on the duration of the measurement period, an individual sample can be measured 20 to 200 times, more preferably 20 to 75 times, and even more preferably 15 to 50 times. As noted above, a major advantage of the screening method of the present invention is that measurements are performed in real time (i.e., multiple measurements of a sample can be performed within a short period of time, thereby providing maximum detection sensitivity, a record of signal changes as a function of time with sufficient time resolution, and ensuring the reliability of the measurements). A single measurement can last between 0.5 and 5 seconds, particularly between 1 and 3 seconds, and more particularly between 1 and 2 seconds. The time interval between two measurements of the same individual sample can be between 1 and 100 seconds, specifically between 1 and 90 seconds, and more specifically between 10 and 80 seconds or between 10 and 60 seconds. When using a microplate consisting of multiple individual samples and a measurement device that measures all microplate wells sequentially (rather than all simultaneously), the time interval between two measurements of the same individual sample will depend on the total number of samples being measured. However, considering that the duration between one measurement and moving to the next sample cannot be shorter than 0.2 seconds in the Tecan Infinite M200 Pro plate reader, the time interval between two measurements of the same individual sample should not be shorter than 20 seconds for a full 96-well microplate or 30 seconds for a 384-well microplate. Based on these data, those skilled in the art will be able to calculate the time interval between two measurements of the same individual sample when other types of plate readers are used. According to one embodiment, between two measurements of every sample, the measured sample is subjected to gentle stirring in order to homogenize its contents. The stirring parameters can be determined by a person skilled in the art based on his general knowledge and / or after carrying out some conventional tests. Among the stirring parameters, it is particularly necessary to determine the shaking mode, amplitude and frequency.

[0052] According to one embodiment, when the sample is in a microplate, the shaking mode can be selected from orbital shaking, linear shaking, horizontal shaking, or XY-axis shaking. The stirring amplitude can be configured to be 0.1 mm to 10 mm, preferably 1 mm to 5 mm, and more preferably about 3 mm. The vibration frequency can be selected from 50 rpm to 500 rpm, preferably 100 rpm to 300 rpm, and more preferably 200 rpm to 250 rpm. In a preferred embodiment of the screening method of the present invention, when the samples are in a microplate, the agitation is performed with the following parameters: shaking mode: orbital, amplitude: 3 mm, frequency: 218.3 rpm. Since the inventors have shown that in the presence of a sufficient concentration of a bactericidal compound, leakage of ATP or its analogues can begin within minutes, step d) is preferably initiated immediately after step c) is performed, i.e., as soon as the candidate composition (or culture medium or known bactericidal compound as some negative and positive controls, respectively) is added to the mixture of bacterial sample, luciferin and thermostable luciferase.

[0053] As is clear from the above, the screening method of the present invention simply involves preparing a bacterial sample (step a), adding further components (steps b) and c), and measuring bioluminescence in real time during incubation of the final sample in step d). Therefore, the method does not require any handling of the bacterial sample that would induce chemical (no harsh dilution in water) or mechanical (no centrifugation) stress on the bacterial sample, or any waiting steps that would degrade ATP or its analogs. This makes the screening method of the present invention faster, more sensitive, and more reliable.

[0054] Step Order First, step a) is performed. Steps b) and c) are performed after step a) and before step d), provided that steps b) and c) are performed between steps a) and d), they may be performed in any order, such as step b) before step c), step c) before step b), or step b) and c) simultaneously (if the mixture of luciferin and thermostable luciferase and the candidate composition are premixed).

[0055] According to a preferred embodiment of the method of the present invention, step c) is performed after or simultaneously with step b) to improve real-time measurement of ATP efflux. Indeed, if step c) is performed before step b), ATP efflux may begin before luciferase / luciferin is added, and as a result, the initial phase of ATP efflux may be missed. More preferably, step c) is performed after step b), preferably 2 to 15 minutes after step b), more preferably 3 to 10 minutes after step b), even more preferably 4 to 6 minutes after step b), and in particular about 5 minutes after step b). This delay between steps b) and c) allows the luciferase / luciferin mixture to consume any traces of ATP that may be present in the bacterial culture and thereby affect the initial reading of the bioluminescence signal during step d).

[0056] Nevertheless, if step c) is performed before step b), step b) should be performed as soon as possible after step c), for example less than 1 minute, preferably less than 30 seconds after step c).

[0057] Negative and positive controls To improve the reliability of the screening method, the screening method preferably also measures bioluminescence in negative and / or positive control samples in real time. Preferably, the method further measures bioluminescence in real time, particularly at the end of step c), for a set of screening samples consisting of the test bacterial sample, the mixture of luciferin and thermostable luciferase, and the candidate composition, for: at least one negative control sample containing a mixture of the test bacterial sample, luciferin, and thermostable luciferase, but not containing the candidate composition or a known bactericidal composition; and / or At least one positive control sample containing a test bacterial sample, a mixture of luciferin and thermostable luciferase, and a known bactericidal composition.

[0058] Since the negative control sample should not contain any bactericidal compounds, no measurement of bioluminescence in the negative control sample is expected, confirming that the experiment is reliable and that any increase in bioluminescence measured in any screening sample containing a candidate composition actually indicates the presence of at least one bactericidal compound in the candidate composition and is not a false positive result. Since the positive control sample is assumed to contain a compound with known bactericidal effect or ATP, and an increase in bioluminescence in the positive control sample is expected, it can be confirmed that the experiment is reliable and that the absence of an increase in bioluminescence measured in the screening sample containing the candidate composition indicates the actual absence of at least one bactericidal compound in the candidate composition and is not a false negative result. Furthermore, if an increase in bioluminescence is measured in both the positive control sample and the screening sample, a comparison of the bioluminescence measured in both samples will provide insight into the bactericidal activity of the candidate composition at the test concentration.

[0059] Implementing screening methods in microplates In a preferred embodiment, the screening method of the present invention is carried out in a microplate, which is particularly practical and allows the method to be easily automated, since there are many devices in the art that allow for the simultaneous addition of additional components to the wells of the microplate, as well as microplate bioluminescence readers. In this embodiment, the microplate may be any microplate classically used in screening methods. In particular, the microplate may be made of plastic or polystyrene. Furthermore, the microplate may be colored white by adding titanium dioxide to improve luminescence detectability. According to a preferred embodiment, the microplate is not black or dark; more preferably, the microplate is light-colored (e.g., light gray, ivory, light beige, etc.), and even more preferably, the microplate is white. The use of a light-colored microplate (preferably white) reflects light, while a dark-colored microplate (e.g., black) absorbs part of the emitted light, which can explain the observed difference in susceptibility based on the final OD600 of the bacteria in the test sample.

[0060] Microplates used in the screening methods of the present invention can have various numbers of wells, such as 6, 12, 24, 48, 96, 384, or 1536 wells, depending on the number of candidate compositions to be screened. The wells can have flat, round, or V-bottoms. The total volume of each well (up to the top of the well) and the recommended working volume (the maximum volume recommended by the manufacturer to prevent spillage and contamination between wells) will vary depending on the number of wells in the microplate. In particular, 96-well microplates generally have recommended working volumes between 25 μL and 200 μL, and 384-well microplates generally have recommended working volumes between 5 μL and 80 μL.

[0061] In most cases, many candidate compositions will be screened, and therefore preferred microplates for use in the screening methods of the present invention have a large number of wells, such as 96 or 384 wells, with recommended working volumes per well of 25 μL to 200 μL and 5 μL to 80 μL, respectively. Preferably, at the end of step c), the microplate comprises: i) at least one screening well containing a test bacterial sample, a mixture of luciferin and thermoluciferase, and a candidate composition; ii) at least one negative control well containing the test bacterial sample, a mixture of luciferin and thermostable luciferase, but no candidate composition or known bactericidal composition; and / or iii) at least one positive control well containing a test bacterial sample, a mixture of luciferin and thermostable luciferase, and a known bactericidal composition or ATP; Includes.

[0062] Preferably, at the end of step c), the microplate contains at least one screening well i) as defined above, at least one negative control well ii) as defined above, and at least one positive control well iii) as defined above. The microplate may also contain multiple wells with the same components. In particular, the same candidate composition may be added to several different wells containing a bacterial sample and a mixture of luciferin and thermostable luciferase to test the bactericidal effect of the candidate composition at various concentrations and / or to replicate measurements (in duplicate or triplicate) at the same concentrations to improve the reliability of the results. According to another embodiment, the screening method of the present invention may also be carried out in a microfluidic tool for ultra-high throughput screening.

[0063] Method for determining the susceptibility of a bacterial sample from a subject suffering from a bacterial infection to a group of known antibiotics (method for determining antibiotic susceptibility) Furthermore, based on the principle of the screening method of the present invention, the present inventors have developed a method for determining the susceptibility of a bacterial sample derived from a subject suffering from a bacterial infection to a group of known antibiotics in real time, with the aim of clinically quickly determining antibiotics that have an efficient bactericidal effect against pathogenic bacteria in the test sample.

[0064] Thus, according to a second aspect, the present invention provides a method for determining the susceptibility of a bacterial sample derived from a subject suffering from a bacterial infection to a group of known antibiotics, comprising: a) inoculating a culture medium with a bacterial sample from a subject suffering from a bacterial infection and, optionally, amplifying the bacteria in the sample; b) dividing the bacterial sample of step a) into a number of subsamples, the number of samples being at least equal to the number of known antibiotics to be tested; c) adding a mixture of luciferin and thermostable luciferase to each subsample; d) spiking one or more subsamples with each of a group of known antibiotics; e) incubating a subsample containing a mixture of luciferin and thermostable luciferase and a known antibiotic at a temperature of 20-60°C (a temperature selected to match the thermostability parameters of the luciferase), preferably 35-37°C, and measuring bioluminescence in real time; Including, the optical density at 600 nm (OD600) of the live bacteria in the subsample after steps a), b), c) and d) have been performed is at least 0.0002; wherein an increase in bioluminescence measured in the sub-sample in step e) indicates that the bacterial sample from the subject is sensitive to the added concentration of the known antibiotic added to the sub-sample in step d).

[0065] Step a): inoculating a culture medium with a bacterial sample from a subject suffering from a bacterial infection and, optionally, amplifying the bacteria in the sample; Step a) of the method for determining antibiotic susceptibility of the present invention is carried out using a previously obtained sample from a subject suffering from a bacterial infection, which may be, in particular, a blood sample, a urine sample, a saliva sample, a fecal sample, a stool sample, a sputum sample, a bronchoalveolar lavage fluid sample, an endotracheal aspirate sample, an oropharyngeal or nasopharyngeal sample, a skin sample, a wound sample, a body fluid (e.g., cerebrospinal fluid, bile fluid, pleural effusion), a vaginal or abdominal abscess secretion, or a tissue sample.

[0066] In step a), the sample is inoculated into a culture medium. Preferably, the culture medium is capable of maintaining the viability of bacterial cells throughout the test. Such media are well known in the art and include, in particular, rich media (e.g., cation-adjusted Mueller-Hinton (MH) and Lysogeny Broth (LB)) and minimal media supplemented with a carbon source such as glucose (e.g., MOPS-glucose), as defined for the screening method of the present invention and disclosed above. Reference media in international guidelines for antibiotic susceptibility testing are unsupplemented cation-adjusted MH broth for testing non-fastidious organisms and MH-F (cation-adjusted MH broth supplemented with 5% mechanically defibrated horse blood and 20 mg / L β-NAD) for testing fastidious organisms. EUCAST provides regularly updated guidelines under the auspices of the European Society for Clinical Microbiology and Infectious Diseases (ESCMID) and the European Centre for Disease Prevention and Control (ECDC).

[0067] Optionally, the bacteria contained in the sample are amplified to reach a sufficient optical density at 600 nm (OD600) to achieve a final OD600 at the end of step d) (after addition of both the luciferin / thermostable luciferase mixture and the known antibiotic) of at least 0.0002, preferably at least 0.0003, at least 0.0005, at least 0.001, at least 0.005, at least 0.01, more preferably at least 0.015, preferably 0.0002-0.5, 0.0003-0.5, 0.0005-0.5, 0.001-0.5, 0.005-0.5, 0.01-0.5, 0.015-0.5, 0.1-0.5, 0.1-0.3, even more preferably 0.1-0.2, especially 0.15, as disclosed herein in the context of the method for screening for bactericidal compounds according to the invention. Preferably, when the volumes added in steps c) and d) result in a dilution factor of about 2 (i.e., the volume after both additions is about twice the initial volume of the subsample in step b), the pathogenic bacteria are amplified to an OD600 of at least 0.0004, at least 0.0006, at least 0.001, at least 0.002, at least 0.01, at least 0.02, or at least 0.03, preferably 0.0004-0.6, 0.0006-0.6, 0.001-0.6, 0.002-0.6, 0.01-0.6, 0.03-0.6, 0.1-0.6, 0.2-0.6, more preferably 0.2-0.4, and most preferably about 0.3. Amplification of bacteria in culture medium can be carried out according to any method known in the art.

[0068] The antibiotic susceptibility determination method as a screening method of the present invention preferably excludes any chemical or mechanical manipulations that may disrupt or damage the physiological functions of pathogenic bacteria in the test sample, i.e., any manipulations that may induce chemical or mechanical stress on live bacteria in the sample. Thus, according to one embodiment of the antibiotic susceptibility determination method of the present invention, the bacteria in the test sample in step a) are not exposed to mechanical (particularly centrifugation) or chemical (particularly osmotic shock) stresses that may damage the bacterial structure and / or bacterial membrane and its function. Mechanical stress and chemical stress are as defined above for the screening method of the present invention.

[0069] In some embodiments of the antibiotic susceptibility determination method of the present invention, even if the type of bacteria present in the bacterial sample is unknown, this does not prevent the antibiotic susceptibility determination method of the present invention from being performed. The bacterial sample can then be tested for susceptibility to several different classes of antibiotics.

[0070] In other embodiments, the type (species or at least genus) of bacteria present in the bacterial sample is known from other analytical methods. This may be advantageous for testing specific classes of antibiotics known in the art to be effective against non-resistant bacteria of the type present in the bacterial sample. In particular, several species and genera of bacteria are known to be particularly associated with bacterial infections in animals, more particularly mammals, including humans, and are therefore of particular medical interest. Therefore, such bacteria can preferably be tested in the antibiotic susceptibility determination method according to the present invention. These include, for example, coagulase-negative Staphylococcus, Mycobacterium tuberculosis, many Streptococcus species (e.g., Streptococcus pneumoniae and Streptococcus pyogenes), Pseudomonas species (e.g., Pseudomonas aeruginosa), Enterococcus faecalis, Escherichia coli, Proteus mirabilis, Serratia marcescens, and Citrobacter freundii. Also of great interest are the ESKAPE group, antibiotic-resistant "priority pathogens" as defined by the World Health Organization (WHO). ESKAPE is an acronym for the following Gram-positive and Gram-negative species: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species. The WHO has published a catalog of 12 bacterial families that pose the greatest threat to human health. These are: Helicobacter pylori (clarithromycin-resistant), Campylobacter (fluoroquinolone-resistant), Salmonella (fluoroquinolone-resistant), Neisseria gonorrhoeae (cephalosporin-resistant, fluoroquinolone-resistant), Streptococcus pneumoniae (penicillin-nonsusceptible), Haemophilus influenzae (ampicillin-resistant), and Shigella species (fluoroquinolone-resistant).

[0071] Step b): Dividing the bacterial sample from step a) into a number of subsamples, with the number of samples being at least equal to the number of known antibiotics to be tested. In step b) of the antibiotic susceptibility determination method of the present invention, the bacterial sample from step a) is divided into several subsamples, the number of which corresponds to at least the number of known antibiotics to be tested. All subsamples preferably contain the same amount of culture medium and have the same OD600 of at least 0.1, preferably 0.15 to 0.5, and more preferably about 0.3.

[0072] Step c): Add a mixture of luciferin and thermostable luciferase to each subsample. The antibiotic susceptibility determination method of the present invention is based on the same principle as the screening method of the present invention, i.e., the conversion of ATP or its analogues leaked by bacteria exposed to a bactericidal compound into bioluminescence by luciferase. Therefore, the antibiotic susceptibility determination method according to the second aspect of the present invention can also use the same thermostable luciferase and luciferin as those described above in the context of the screening method of the present invention under the same conditions. According to one embodiment of the sensitive detection method of the invention, before step d), a short pre-incubation step (3 to 10 minutes, e.g. 3, 4, 5, 6, 7, 8 minutes, in particular 4 to 6 minutes, preferably 5 minutes) is carried out in order to gradually reach the incubation temperature and to remove any extracellular ATP that may already be present (by conversion by luciferase before the addition of the antibiotic).

[0073] Step d): spiking at least one subsample with each of a group of known antibiotics. After adding luciferin / thermostable luciferase in step c), in step d) each antibiotic from the group of known antibiotics tested is added to at least one subsample to determine the susceptibility of the bacterial sample. Any antibiotic known to have a bactericidal effect (i.e., susceptibility to killing bacteria) on at least some bacteria (i.e., non-resistant bacteria) can be tested for susceptibility and thus added to at least one subsample in step d). Preferably, the antibiotic tested for susceptibility is a known bactericidal antibiotic selected from the group consisting of: Aminoglycosides (streptomycin, kanamycin, gentamicin, neomycin, amikacin, tobramycin, netilmicin, etc.). Beta-lactams, including penams (such as penicillin, amoxicillin, and ampicillin), carbapenams, clavams (such as clavulanic acid), penems, carbapenams, cephems (such as cephalexin and cefpodoxime), carbacephems, oxacephems, and monobactams. Quinolones, including fluoroquinolones (such as ofloxacin, levofloxacin, and ciprofloxacin). Polypeptides (such as polymyxin B); glycopeptides and lipoglycopeptides (such as vancomycin, teicoplanin, oritavancin, dalbavancin, and telavancin). Trimethoprim alone or in combination with sulfamethoxazole Trimethoprim is bactericidal in rich media, and results with this antibiotic should be analyzed with caution, as the inventors found that at concentrations above 200 μg / mL, this drug inhibited the luciferase reaction. Oxazolidinones, linezolid (usually classified as a bacteriostatic agent but may also have bactericidal properties), tedizolid. Among macrolides, erythromycin, telithromycin, azithromycin, and roxithromycin have been reported to have bactericidal and bacteriostatic effects.

[0074] A preferred list of known bactericidal antibiotics from which the susceptibility of a bacterial sample to be determined by the antibiotic susceptibility determination method of the present invention is selected includes: amoxicillin, amoxicillin-clavulanate, ampicillin, ticarcillin, piperacillin-tazobactam, ertapenem, cefoxitin, cefixime, ceftriaxone, cefotaxime, ceftazidime, gentamicin, amikacin, nalidixic acid, ofloxacin, ciprofloxacin, fosfomycin and trimethoprim-sulfamethoxazole.

[0075] According to one embodiment, the susceptibility determination method of the present invention may also be carried out to determine the susceptibility of known combinations of antibiotics, for example, antibiotic combinations that correspond to antibiotics that are typically used in combination (administered simultaneously or sequentially) to treat diseases caused by one or more bacteria.

[0076] Although less preferred, the antibiotic susceptibility detection method of the present invention can also be used to test antibiotics known to have bacteriostatic effects on at least some bacteria (i.e., antibiotics that inhibit bacterial growth and proliferation without killing the bacteria). Indeed, data obtained by the present inventors indicates that bacteria do not leak ATP or analogs in the presence of bacteriostatic compounds, although some compounds may be bacteriostatic at low concentrations and bactericidal at high concentrations. Antibiotics known to be bacteriostatic for at least some bacteria may be bactericidal for other bacterial strains or at high concentrations. Such antibiotics, primarily known as bacteriostatic agents, that can be tested in the antibiotic susceptibility determination method of the present invention may be selected from the group consisting of macrolides (e.g., azithromycin, erythromycin, telithromycin, roxithromycin), oxazolidinones (e.g., linezolid), sulfonamides, and tetracyclines (e.g., doxycycline).

[0077] At the end of step d) (i.e., after all components of the reaction have been added), the final concentration of the tested antibiotic in each subsample is preferably between 0.002 μg / mL and 1024 μg / mL, as determined by two-fold serial dilutions (20 values). For simplicity, the concentration range obtained by the two-fold serial dilutions can be 1 / 500 to 1000 / 1, preferably 1 / 4 to 4 / 1, of the MIC of the known antibiotic against a known susceptible bacterial strain in the culture medium used, and is preferably less than 1024 μg / mL. The MICs of known antibiotics in various culture media are well known in the art or can be easily determined by routine experimentation by those skilled in the art.

[0078] Preferably, at the end of step d) (i.e. after all components of the reaction have been added): The OD600 of the live bacteria is at least 0.1, preferably 0.1 to 0.3, more preferably 0.1 to 0.2, and especially 0.15. The luciferin solution is diluted to the concentration recommended by the luciferin manufacturer or to the optimal concentration based on preliminary experiments (6-9 times when using Kikkoman's "CheckLite AT100" kit or NanoLuc® (NLuc)); The thermostable luciferin solution is diluted to the concentration recommended by the luciferin manufacturer or to the optimal concentration based on preliminary experiments (6-9 times when using Kikkoman's "CheckLite AT100" kit or NanoLuc® (NLuc)); and / or The concentration of the known antibiotic is 0.002 μg / mL to 1024 μg / mL, preferably 1 / 4 to 4 / 1 of the MIC of the known antibiotic against known susceptible bacterial strains in the culture medium used, and is preferably maintained below 1024 μg / mL. The dilution values ​​of the luciferin and luciferase solutions mentioned above can be adjusted according to the instructions provided with the luciferin / luciferase kit.

[0079] Step e): Incubate the subsample under stirring at a temperature of 20 to 60 ° C, preferably 35 to 37 ° C, and measure bioluminescence in real time (measurement step). Measurement step e) of the antibiotic susceptibility determination method of the present invention, which involves incubating a subsample under stirring at 20 to 60°C (a temperature selected consistent with the thermostability parameters of luciferase), preferably 30 to 40°C, more preferably 35 to 37°C, and measuring bioluminescence in real time, corresponds to measurement step d) of the screening method of the present invention.

[0080] Therefore, all technical features and technical definitions provided above for the measuring step d) of the screening method can be applied to the measuring step e) of the antibiotic susceptibility determination method of the present invention. In particular, bioluminescence is measured in real time as described above, and an increase in bioluminescence measured in the subsample indicates that the bacterial sample from the subject is sensitive to the spiked concentration of the known antibiotic spiked into the subsample in step c).

[0081] Step Order Step a) is performed first and step b) is performed second. Steps c) and d) are carried out after steps a) and b) and before step e), provided that they are carried out between steps b) and e), steps c) and d) may be carried out in any order, such as step c) before step d), step d) before step c), or both steps c) and d) simultaneously (if the mixture of luciferin, thermostable luciferase, and known antibiotic is premixed).

[0082] According to a preferred embodiment of the method of the present invention, step d) is performed after or simultaneously with step c) to improve real-time measurement of ATP efflux. Indeed, if step d) is performed before step c), ATP efflux may begin before luciferase / luciferin is added, resulting in the possibility of missing the initial phase of ATP efflux. Preferably, step d) is performed simultaneously with or after step c). More preferably, step d) is performed after step c), preferably 2 to 15 minutes after step c), more preferably 3 to 10 minutes after step c), even more preferably 4 to 6 minutes after step c), and in particular about 5 minutes after step c). This delay between steps c) and d) allows the luciferase / luciferin mixture to consume any traces of ATP that may be present in the bacterial culture and thereby affect the initial reading of the bioluminescence signal during step d). Nevertheless, if step d) is performed before step c), step c) should be performed as soon as possible after step d), for example less than 1 minute, preferably less than 30 seconds after step d).

[0083] Negative and positive controls To improve the reliability of the antibiotic susceptibility determination method, it is preferred that the antibiotic susceptibility determination method also measures bioluminescence in the negative and / or positive control samples in real time. The first type of negative control sample is a sample containing culture medium, a mixture of luciferin and thermostable luciferase, and a known antibiotic instead of the bacterial subsample at the end of step d) (i.e., immediately before incubation and real-time bioluminescence measurement). Indeed, in the absence of a bacterial sample, no bioluminescence can be expected. The second type of negative control sample is a sample containing a bacterial subsample, culture medium instead of the luciferin and thermostable luciferase mixture, and a known antibiotic at the end of step d) (i.e., immediately before incubation and real-time bioluminescence measurement). Indeed, in the absence of the luciferin and thermostable luciferase mixture, no bioluminescence can be expected. A third type of negative control sample is a sample containing a bacterial subsample, a mixture of luciferin and thermostable luciferase, and culture medium instead of a known antibiotic at the end of step d) (i.e., immediately before incubation and real-time bioluminescence measurement). Indeed, in the absence of antibiotics, no bioluminescence can be expected. Because the susceptibility of the bacterial sample to known antibiotics is unknown, a traditional positive control (using a compound known to be bactericidal against the bacteria) is not possible. Instead, an alternative positive control, such as pure ATP, can be used. In this case, ATP is added instead of the antibiotic, and bioluminescence measurements are taken immediately thereafter. Those skilled in the art will be able to determine the concentration of ATP to use from the manufacturer's guidelines (e.g., in the range of 5–50 nM).

[0084] Implementation of a microplate-based antibiotic susceptibility determination method Furthermore, the antibiotic susceptibility determination method of the present invention is preferably carried out using a microplate, which may have any of the features or combination of features described above in the section on screening methods.

[0085] In this case, in step b) of the antibiotic susceptibility determination method of the present invention, the bacterial sample of step a) is divided into several subsamples, the number of which corresponds at least to the number of known antibiotics to be tested, and which are dispensed into several different wells of a microplate. Again, all subsamples preferably contain the same volume of culture medium and have the same OD (calculated so that the final OD after all components of the reaction have been added is at least 0.0002, preferably at least 0.0003, at least 0.0005, at least 0.001, at least 0.005, at least 0.01, more preferably at least 0.015, more preferably 0.0002-0.5, 0.0003-0.5, 0.0005-0.5, 0.001-0.5, 0.005-0.5, 0.01-0.5, 0.015-0.5, 0.1-0.5, 0.1-0.3, even more preferably 0.1-0.2, especially about 0.15). To provide a first type of negative control, some wells of the microplate can be dispensed with culture medium without bacteria.

[0086] In step c), a mixture of luciferin and thermostable luciferase is dispensed into the wells of the microplate containing the bacterial subsamples and, optionally, into the wells of the microplate containing culture medium without bacteria. To have a second negative control, culture medium can be dispensed into some wells of the microplate into which the bacterial subsamples were previously dispensed, instead of the mixture of luciferin and thermostable luciferase.

[0087] In step d), the antibiotic to which the bacterial sample's susceptibility is tested is added to different microplate wells. The same antibiotic may be added to multiple different wells to test the bacterial sample's susceptibility to different concentrations of the antibiotic and / or to replicate measurements (in duplicate or triplicate) to improve the reliability of the results. Similarly, several wells with the same negative control may be present within the microplate (e.g., in duplicate or triplicate). To have a third type of negative control, culture medium may be dispensed instead of a known antibiotic into some wells of a microplate that has previously dispensed a bacterial subsample and a mixture of luciferin and thermostable luciferase. The microplate may contain one or more wells consisting of positive control samples, as disclosed above.

[0088] Method for evaluating the minimum inhibitory concentration (MIC) of bactericidal compounds (MIC evaluation method) The present inventors have also demonstrated that by monitoring the reaction of luciferase with ATP or its analogues in real time, while varying the antibiotic concentration, and measuring the time lag between the addition of the antibiotic and the detection of ATP efflux, it is possible to evaluate the minimum inhibitory concentration (MIC) of a compound for a given bacterial sample. In the context of the present invention, the term "minimum inhibitory concentration (MIC)" means the lowest concentration of an antimicrobial compound capable of inhibiting visible growth of a microorganism after overnight incubation. Thus, according to a third aspect, the present invention relates to 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 having an optical density at 600 nM (OD600) of at least 0.0002; b) dividing the bacterial sample of step a) into several subsamples; c) adding a mixture of luciferin and thermostable luciferase to the subsample; d) adding various concentrations of a bactericidal compound to the sub-samples; e) incubating the sub-sample to which the mixture of luciferin and thermostable luciferase and the bactericidal compound has been added at 20-60°C (a temperature selected consistent with the thermostability parameters of the luciferase), preferably 35-37°C, and measuring bioluminescence in real time, wherein the optical density at 600 nm (OD600) of the live bacteria in the sub-sample after steps a), b), c) and d) has been performed is at least 0.0002, preferably 0.15; f) for each concentration of bactericidal compound tested, determining the lag time between the time the bactericidal compound is added and the time an increase in bioluminescence signal is detected; g) expressing lag time as a function of bactericidal compound concentration; h) constructing an exponential decay curve fitted to the lag time measurement points as a function of bactericidal compound concentration; i) determining the lowest bactericidal compound concentration at the plateau of the exponential decay fitting curve; j) determining the lag time amplitude of the exponential decay fitting curve; k) assessing the MIC, wherein the MIC is: the antibiotic concentration corresponding to a lag time equal to (lag time at plateau + 0.3 × lag time amplitude) on the exponential decay fitting curve; The lowest bactericidal compound concentration at the plateau of the exponential decay fitting curve and be evaluated as being included between

[0089] Steps a) to e) Steps a) to e) of the MIC evaluation method of the present invention correspond to steps a) to e) of the antibiotic susceptibility determination method of the present invention and are carried out in the same manner. In particular, in the MIC evaluation method of the present invention, measurement step e) is preferably initiated immediately after step d), i.e., immediately after the bactericidal compound is added to the mixture of the bacterial subsample, luciferin, and thermostable luciferase, so that the lag time between the time when the bactericidal compound is added and the time when an increase in the bioluminescence signal is detected can be determined. Step a) is performed first and step b) is performed second. Steps c) and d) are carried out after steps a) and b) and before step e), provided that they are carried out between steps b) and e), steps c) and d) may be carried out in any order, such as carrying out step c) before step d), step d) before step c), or both steps c) and d) simultaneously (if the mixture of luciferin, thermostable luciferase, and known antibiotic is premixed).

[0090] According to a preferred embodiment of the method of the present invention, step d) is performed after or simultaneously with step c) to improve real-time measurement of ATP efflux. Indeed, if step d) is performed before step c), ATP efflux may begin before luciferase / luciferin is added, resulting in missed early stages of ATP efflux. Preferably, step d) is performed simultaneously with or after step c). More preferably, step d) is performed after step c), preferably 2 to 15 minutes after step c), more preferably 3 to 10 minutes after step c), even more preferably 4 to 6 minutes after step c), and particularly about 5 minutes after step c). This delay between steps c) and d) allows the luciferase / luciferin mixture to consume any traces of ATP that may be present in the bacterial culture and thereby affect the initial reading of the bioluminescence signal during step d).

[0091] Nevertheless, if step d) is performed before step c), step c) should be performed as soon as possible after step d), for example less than 1 minute, preferably less than 30 seconds after step d).

[0092] Step f): For each concentration of bactericidal compound tested, determine the lag time between the time the bactericidal compound is added and the time an increase in bioluminescence signal is detected. In the context of the present invention, the term "lag time" relates to the period between two related events, here the time from the addition of the bactericidal compound to the first detection of an increase in bioluminescence. We observed that there was a delay between the addition of the bactericidal compound and the first detectable increase in bioluminescence due to luciferase-mediated conversion of leaked ATP or its analogs, and that this delay was shorter with increasing concentrations of the bactericidal compound. The time of addition of the bactericidal compound is known. In step e), bioluminescence is measured for various concentrations of the same bactericidal compound, and the bioluminescence curves obtained for each concentration are analyzed to determine the time at which an increase in bioluminescence is first detected in each curve. In the bioluminescence curve, this time corresponds to the first time point in the kinetics where the signal increases compared to the previous time point and this increase continues, indicating a sudden increase due to the effect of the bactericidal antibiotic. More precisely, since in some cases the baseline may increase slightly at the beginning of the kinetics, the first increase should be considered as the main characteristic peak. This time point can be identified by visual inspection or mathematical analysis. Curve analysis can be performed by noise removal (Fourier transformation, exponential smoothing, moving average, etc.) to accurately define the delay. Such analysis can be performed, for example, using Microsoft Excel®. The lag time obtained for each concentration of bactericidal compound tested is then calculated by subtracting the time the bactericidal compound was added from the time the first increase in bioluminescence was detected.

[0093] Step g): Depicting lag time as a function of the concentration of the bactericidal compound In step g), the lag time obtained by varying the test concentration of the bactericidal compound can be represented schematically as a function of the bactericidal concentration tested. A graph is created with the concentration of the added bactericidal compound (μg / mL) on the x-axis and the lag time determined in step f) on the y-axis. The graph should contain as many experimental points as there are concentrations of the bactericidal compound tested.

[0094] Step h): Constructing an exponential decay curve to fit the lag time measurement points as a function of the bactericidal compound concentration. For all tested bactericidal compounds (see the Examples, especially Figures 22-23), the inventors found that the various points on the graphs representing lag time as a function of the concentration of the tested bactericide could be fitted by an exponential decay curve. Specifically, the x and y coordinates of each experimental point can be entered into software such as GraphPad Prism or Microsoft Excel®, and a single exponential decay fit function can be selected to perform a fit. Parameters that indicate the quality of the fit must be determined. As a result of the fit, the signal plateau value and lag time amplitude (span) can be accessed. These parameters are necessary for MIC evaluation.

[0095] Step i): Determining the lowest bactericidal compound concentration at the plateau of the exponential decay fitting curve The plateau indicates the limit at which further increases in the concentration of the bactericidal compound do not further decrease the lag time. In an exponential decay curve, the plateau corresponds to the horizontal portion of the curve. The plateau is described as a result parameter of the fit. One or more of the experimental points may be located in the plateau portion of the exponential decay curve.If only one experimental point is located in the plateau portion of the exponential decay curve, the bactericidal compound concentration at this point is taken as the lowest bactericidal compound concentration in the plateau portion of the exponential decay fitting curve.If multiple experimental points are located in the plateau portion of the exponential decay curve, the point with the lowest bactericidal compound concentration is selected, and the bactericidal compound concentration at this point is taken as the lowest bactericidal compound concentration in the plateau portion of the exponential decay fitting curve.

[0096] Step j): Determining the lag time amplitude of the exponential decay fitting curve The "lag time amplitude" is defined as the difference between the lag time value at the y-axis intercept of the exponential decay fit curve and the lag time value at the plateau of the exponential decay fit curve. The lag time amplitude (span) is listed in the fit result parameters.

[0097] Step k): Evaluation of MIC concentration In this final step, the MIC concentration of the bactericidal compound is determined by the antibiotic concentration corresponding to a lag time equal to (lag time at plateau + 0.3 × lag time amplitude) on the exponential decay fitting curve; the lowest bactericidal compound concentration at the plateau of the exponential decay fitting curve determined in step i) above; is evaluated as being contained between [Example]

[0098] Materials and Methods Bacterial strains and growth conditions Escherichia coli MG1655 and Bacillus luteus were used for antibiotic susceptibility testing. Cultures were performed at 37°C in rich Luria broth (LB) (10 g NaCl, 10 g tryptone, 5 g yeast extract / L) or MOPS minimal medium supplemented with 0.4% glucose. Staphylococcus aureus USA300 was cultured in tryptic soy broth (supplemented with 0.25% glucose) at 37°C.

[0099] Antibiotics and Reagents Neomycin (N6386), apramycin (A2024), azithromycin (PHR1088), kanamycin (K1377), spectinomycin (S4014), ampicillin (A9518), tetracycline (T7660), streptomycin (S9137), cephalexin (C4895), polymyxin B (P4932), erythromycin (E6376), puromycin (P7255), ofloxacin ( O8757), amoxicillin (31586), amikacin (PHR1654), cefixime (CDS021590), cefoxitin (C4786), ceftazidime (CDS020667), ceftriaxone (C5793), ciprofloxacin (PHR1167), fosfomycin (P5396), nitrofurantoin (46502), piperacillin (93129), and sulfamefoxazole (31737) were purchased from Sigma-Aldrich. Chloramphenicol (018043) was purchased from Eurobio. 3-(N-morpholino)propanesulfonic acid (MOPS, M3183) was purchased from Sigma-Aldrich. Glucose (24379.294) was obtained from VWR BDH Chemicals. Bacto™ Tryptone (211705) and Bacto™ Yeast Extract were purchased from BD Biosciences Advanced Bioprocessing, and sodium chloride (06404.1000) was purchased from Merck. Bacto Tryptic (Trypic) Soy Broth (Ref 0370-17-3) was purchased from Difco.

[0100] Real-time bioluminescence assay Classical luciferase was used from Molecular Probes (ATP Determination Kit (A22066)). Thermostable luciferase was used from "CheckLite AT100" (Kikkoman). Luciferin-luciferase reagent was prepared according to the manufacturer's instructions. Exponentially growing bacteria (OD ) from the culture medium were added to each well (60 μl / well) of a black 96-well microtiter plate (Greiner Bio-one, 655076). 600 A 100-μL (pH 7.5) solution containing 100 μL of luciferin-luciferase reagent solution was added to the wells. The microtiter plate was incubated at 37°C for 5 minutes to allow luciferase to react with the luciferase and eliminate traces of extracellular ATP. 40 μL of a 3x concentrated antibiotic solution prepared in culture medium was then added. Bioluminescence in each well was monitored for 4 hours under shaking (218 rpm) at 28°C or 37°C, depending on the experiment (see figure legend). The stability of the reagent in the presence or absence of antibiotic was confirmed in a control experiment containing no bacteria but 4 pmol of ATP. Tests were also performed in a 384-well microtiter plate (Greiner Bio-one, 784076).

[0101] Detection of commercial bactericidal compounds in supernatants The ATP measurement protocol developed by the inventors was adjusted and optimized to increase the sensitivity of the test. The same kit, "CheckLite AT100," which uses firefly luciferin and luciferase, was used. Exponentially grown culture medium (OD) in minimal medium (MOPS-glucose 0.4%) 600Bacteria in a 1000-well plate (pH = 0.6) were used. Sixty microliters of the bacterial cell suspension was added to each well of a black microtiter plate (black walls and black bottom), and 10 μL of luciferin-luciferase reagent solution was added to each well. To eliminate the possibility of traces of extracellular ATP released by the bacteria, the plate was incubated at 37°C for 5 minutes with shaking (218 rpm). Then, 20 μL of Streptomyces fradiae supernatant (stored on ice during incubation) was added to each well. Bioluminescence from each well was monitored for 4 hours at 37°C with shaking (218 rpm) using a microtiter plate reader (InfinitePro200, TECAN).

[0102] Resistant bacterial strains and biofilms Biofilms were prepared according to standard protocols. Briefly, S. aureus USA300 was incubated overnight in tryptic soy broth (containing 0.25% glucose) at 37°C and 180 rpm. Pre-sterilized wells of a FLUOTRAC™ microplate were filled with 10% ethanol at an average concentration of 10%. 5 ~10 6 150 μl of inoculated broth diluted to CFU / ml was filled into the well. The microplate was covered and incubated at 37°C for 18 hours. The biofilms were then washed twice with 150 μl of MOPS. 80 μl of the wash solution was not discarded but added to an adjacent well to observe the amount of ATP contained. Next, the optimized protocol developed by the inventors for the bioluminescence assay was followed.

[0103] Antibiotic MIC measurement The minimum inhibitory concentration (MIC) of antibiotics was measured using an ATP assay kit under the same culture conditions as the test. Two-fold serial dilutions of the test antibiotics were prepared in LB or MOPS glucose 0.4% medium. Exponentially growing cultures (OD ) were added to each well (60 μl / well) of a sterile microtiter plate containing the test concentration of antibiotic (40 μl / well). 600The volume was adjusted to 120 μl with medium, and the OD was measured using a microtiter plate reader (Infinite 200 PRO, TECAN) for 17 hours at 37°C. 600 nm measurements were carried out.

[0104] result Bioluminescence assay for detecting ATP leakage in real time The principle of this assay is shown in Figure 1.

[0105] We first evaluated the feasibility of detecting ATP in rich and minimal media using classical firefly luciferase. We confirmed that adding ATP to rich Luria Broth (LB) or minimal MOPS media containing the luciferin / luciferase reagent induced luminescence. The reagent's stability was superior in LB media (Figure 2a). In MOPS media, the reagent was rapidly inactivated upon preincubation with the media (Figure 2a). Next, we evaluated the response of live reporter E. coli cells after antibiotic shock with neomycin, an aminoglycoside known to cause leakage of small molecules (amino acids, nucleotides, and potassium). Luminescence measurements demonstrated that this assay reported antibiotic-dependent ATP leakage in real time (Figure 2b). We also evaluated the performance of this assay in rich (LB) or minimal (MOPS) media. In minimal medium, as observed in Figure 2a, no luminescence signal was detected due to the instability of one or several reagents (Figure 2b). First, we concluded that this result indicated that some components of the rich growth medium likely acted as stabilizers of luciferase in the bioluminescence assay. However, because this assay was performed at 28°C (lower than the 37°C typically used for bacterial growth), we repeated the assay at 37°C. The results obtained were unsatisfactory because classical luciferase is less stable in rich and minimal media at 37°C, resulting in a rapid decline in its activity. Control samples without added antibiotics did not appear to provide a signal indicating that ATP release was dependent on the antibiotic (Figure 2b).

[0106] To investigate whether improving the stability of firefly luciferase would improve measurements in minimal and rich media, we performed the same assay using a thermostable firefly luciferase (Figure 2c). Antibiotic-dependent luminescence signals could be recorded in both minimal and rich media, establishing conditions for a real-time ATP assay in live E. coli (Figure 2c). Because ATP, luciferin, and luciferase produce light immediately upon contact, this assay reports the efflux of ATP (or analogs) across the outer membrane in real time. We noted superior performance of the thermostable luciferase in minimal medium (MOPS) compared to rich medium (LB) (Figure 2d). Because the thermostable luciferase can be tested in both rich and minimal media, we pursued further development of the method using this enzyme. The present inventors performed comparative assays to confirm the results obtained with conventional luciferase and thermostable luciferase.

[0107] To perform this assay, classical luciferase (firefly luciferase, Molecular Probes ATP Assay Kit, A22066) and thermostable luciferase (CheckLite kit, Kikkoman, AAE43251.1) were used according to the manufacturer's instructions. Both types of luciferase were added to nutrient-rich LB or minimal MOPS-glucose medium preheated to 37°C. At various times during incubation, aliquots of the solution were withdrawn, replenished with reagent, and mixed with 0.8 pmoles of ATP for bioluminescence readings. Immediately after mixing, bioluminescence measurements were performed at 37°C in a total volume of 12 μL in a 384-well microtiter plate (Greiner Bio-one, 784076) using an InfinitePro200 TECAN plate reader.

[0108] The results shown in Figure 24 confirm that classical firefly luciferase is highly unstable at 37°C in both rich (LB) and minimal (MOPS-G) media. In LB media, the enzyme lost 70% of its activity after only 12 minutes of incubation. In minimal MOPS-G media, the enzyme lost more than 80% of its activity after 30 minutes. These results indicate that classical (non-thermostable) luciferases are not suitable for performing real-time assays to detect ATP efflux at 37°C, the temperature most suitable for bacterial growth.

[0109] On the other hand, when a similar assay was performed using thermostable luciferase (Kikkoman), this enzyme retained 50% of its activity after 5 hours of incubation at 37°C in LB and 90% of its activity after 7 hours of incubation in MOPS-G (Figure 24). In MOPS-G, it retained more than 80% of its activity even after 20 hours of incubation, demonstrating that it is an extremely stable enzyme.

[0110] From these results, we conclude that thermostable luciferase is the only enzyme capable of performing a sensitive and reliable real-time ATP leakage assay at 37°C, the optimal temperature for growth of most bacterial species. The amount of reporter cells (bacteria in the test sample) is important for the assay. The bioluminescence signal was found to depend on the concentration of reporter cells. Reducing the amount of bacteria reduced the bioluminescence signal (Figure 3). The amplitude of the signal also varied as a function of the OD of the cells. We first investigated the effect of low cell amounts (OD 600 = 0.0015 and 0.015), we found that bioluminescence signals were generated at low drug concentrations, but these signals only increased modestly with drug concentration. 600 A value of 0.15 was chosen for the assay because it gives a signal with a large amplitude.

[0111] Therefore, we tested the screening method of the present invention by varying the amount of reporter cells in cation-adjusted Mueller-Hinton medium, which is commonly used in clinical AST. The sensitivity of the method using white microtiter plates improved, reaching the inoculation volume recommended by EUCAST (Figure 25). Cells were grown in Mueller-Hinton medium to an OD of 0.6 and then diluted in Mueller-Hinton medium to an OD of 0.00055. Luciferin-luciferase was then added, followed by antibiotics just before use in tATP bioluminescence measurements (60 μL of bacteria at OD of 0.00055 + 20 μL of luciferin-luciferase reagent + 40 μL of antibiotic). The final OD of the bacteria was 0.000275. The assay was performed at various drug concentrations. White 96-well microtiter plates (Greiner Bio-one, 655075) were used. To improve the accuracy of lag time identification, especially under weak signal conditions, short-term fluctuations in the ATP leakage trace were smoothed using a five-point moving average. This facilitated the identification of the lag time before the onset of ATP leakage, which is necessary for MIC estimation. Analysis of the lag time yielded MIC estimates similar to those obtained from standard growth assays in this medium (Figure 25).

[0112] Trace amounts of extracellular ATP were detected in the cell culture medium during a 5-minute preincubation period before drug addition and were consumed by luciferase. We found that there was no need to wash the cells by centrifugation to replace the culture medium with fresh medium. In fact, we demonstrated that centrifugation and subsequent cell manipulation can promote the uptake of aminoglycoside antibiotics (Figure 4A). Therefore, manipulation of reporter cells should be avoided to prevent bias in the results. We also performed assays in which we allowed bacteria to recover from the stress of centrifugation by adding a drug (neomycin) with or without centrifugation, and only after various time points (a few minutes, 5 minutes, 15 minutes, 30 minutes, etc., up to 120 minutes) after centrifugation. We found that it took approximately 2 hours (120 minutes) after centrifugation for the bactericidal effect of neomycin to return to the same level as in the non-centrifuged sample (Figure 4B).

[0113] We confirmed that the presence of the different antibiotics tested did not affect the activity of the bioluminescence assay (Figure 5). Therefore, bioluminescence was measured (in the absence of bacteria) after preincubation of the luciferase-luciferin reagent with the maximum antibiotic concentration used in this study in LB before adding 30 nM ATP. For puromycin (Figure 5), an adenosine-containing 3'-terminal analog of aminoacylated tRNA, significant inhibition of bioluminescence was observed, but only at concentrations above the minimum inhibitory concentration (MIC) measured in E. coli (Figure 5 and Table 1). Similar observations were made for actinomycin D, novobiocin, and trimethoprim.

[0114] [Table 1]

[0115] After optimization, the amount of reagents used for the bioluminescence assay was reduced to less than 17% of the total volume, minimizing potential perturbations to cell viability. Indeed, we demonstrated that cells were viable under these experimental conditions (Figure 6). Finally, we performed an ATP leakage assay using an aminoglycoside-resistant E. coli strain. Expression of the aminoglycoside O-phosphotransferase APH(3')-IIa prevented detection of ATP leakage upon neomycin addition, indicating that drug-induced ATP leakage was due to the drug's bactericidal activity against bacteria (Figure 7). This further demonstrates that this method can detect the susceptibility / resistance of specific bacterial samples using low concentrations of antibiotics (at or below the MIC). Having obtained a highly sensitive bioluminescence assay that reports ATP leakage from live bacteria (Figure 1), we set out to examine their response after shock to various antibiotics.

[0116] ATP leakage triggered by bactericidal antibiotics is a key signature In all the following experiments, temperature, OD 600 The parameters, such as temperature and equipment, were the same as those used in the bioluminescence assay, and the MIC values ​​of each antibiotic were measured.

[0117] Aminoglycosides are bactericidal antibiotics that cause translational miscoding, resulting in lethal effects on cells. The leakage of small molecules following aminoglycoside shock has been confirmed by various methods, including radiolabeling. Therefore, we investigated the response of living cells to aminoglycoside stress. OD 600Rapidly growing cells with a pH of 0.3 (final pH 0.15) were briefly placed in a plate reader for thermal equilibration, and antibiotics were added at different concentrations around the MIC in LB medium. Increasing the concentration of neomycin revealed a rapid and strong initial ATP release at concentrations above the MIC, revealing a biphasic signal (Figure 8). Under these conditions, leakage occurred within minutes of antibiotic addition, consistent with previously reported leakage of amino acids, nucleotides, and K+. Because the MIC value does not report cell viability, we performed a viability assay with varying neomycin concentrations (Figure 8). The number of colony-forming units (CFUs) remained constant over the first 90 minutes, demonstrating that cells survived neomycin treatment during this period. Therefore, we concluded that the observed biphasic ATP efflux was from still-viable cells. Similar biphasic traces were also observed for five other aminoglycosides: streptomycin, kanamycin, gentamicin, apramycin, and amikacin (Figure 8). We noted a decrease in signal intensity at or near the MIC concentrations for all six aminoglycosides, which correlated with changes in the kinetics of ATP release. Furthermore, the time lag to detect luminescence decreased with increasing antibiotic concentration for all six aminoglycoside antibiotics tested (Table 2).

[0118] [Table 2] TIFF0007795745000003.tif216153 TIFF0007795745000004.tif200153

[0119] At the highest concentrations, the lag time was shortened to approximately 9.4 minutes for neomycin, 4.7 minutes for kanamycin, 10 minutes for streptomycin, and 10.9 minutes for apramycin, which is impossible to achieve. Below the MIC, results should be analyzed carefully. The long lag and weak signal suggest that not all cells accumulated the antibiotic immediately upon drug addition. Furthermore, at drug concentrations below the MIC, cells continued to grow during the test, and the traces may be slightly affected by residual growth. At concentrations above the MIC, the responses were clear and similar, indicating that most cells were affected very quickly after drug exposure (Figure 8). The characteristic features observed with these miscoding agents, i.e., an initial rapid and strong burst followed by a slower but larger phase, were clearly discernible at the MIC concentration.

[0120] Ampicillin is a bactericidal antibiotic of the β-lactam class that rapidly inhibits cell wall synthesis, resulting in slow cell lysis. Addition of increasing concentrations of ampicillin shortened the lag time to ATP release to 9 minutes. Similarly, miscoding agents produced strong and distinct signatures at concentrations above their MIC (25 μg / mL) (Figure 8). Here, ATP release was polyphasic, with an initial phase of very low intensity followed by a second phase of approximately 2 hours with much greater amplitude.

[0121] Polymyxins are bactericidal antibiotics that act primarily on Gram-negative bacteria by altering membrane permeability. Polymyxin B induced a clear, strong, biphasic response even at half the MIC (4 μg / mL). After drug addition, a strong, rapid initial phase was observed, followed by a second phase of large amplitude. At concentrations higher than 4 μg / mL, ATP release began immediately after polymyxin B addition (the lag was shorter than 2.4 min and was not measurable) (Figure 8). Interestingly, the amplitude of the initial burst was maximal at a dose of 4 μg / mL and gradually decreased at higher concentrations (Figure 8). Conversely, the amplitude of the second phase increased with concentration (Figure 8).

[0122] Addition of cephalexin, a cephalosporin-class beta-lactam bactericidal antibiotic, induced a multiphasic response (Figure 8). At subinhibitory concentrations of 8.8 and 17.5 μg / mL, three synchronized phases occurred between 50 and 250 min. At 35 μg / mL, a burst of increasing amplitude was detected with increasing concentration, reminiscent of the initial burst observed with miscoding agents such as ampicillin and polymyxin B. Unlike polymyxin B, the amplitude of the initial burst increased in a concentration-dependent manner after a lag of 3.6 min (Figure 8), which could not be further shortened. The amplitudes of the three subsequent synchronized phases varied between 50 and 250 min.

[0123] Ofloxacin belongs to the fluoroquinolone class and inhibits bacterial DNA gyrase, inhibiting DNA replication in growing bacteria. It is a bactericidal antibiotic. Its effect on ATP release was rather slow, with efflux observed after 77 min at the highest concentration tested (Figure 8 and Table 2). ATP release appeared monophasic, and its amplitude varied depending on the concentration of the drug used. The data for the bactericidal antibiotics tested are summarized in Figure 10 A. In conclusion, for all bactericidal antibiotics tested, we observed monophasic or multiphasic ATP leakage. These data were confirmed by performing novel assays following the same experimental conditions as those disclosed above. The results of these assays are summarized in Figure 10B and confirm the ability of the method according to the invention to rapidly identify bactericidal compounds.

[0124] Bacteriostatic antibiotics have a weak effect on promoting ATP leakage Next, we examined several bacteriostatic antibiotics that inhibit protein synthesis. The ribosome-targeting antibiotics erythromycin and spectinomycin induced very weak amplitudes of ATP release, which are incomparable to what we observed with bactericidal drugs (Figures 9 and 10). For tetracycline and azithromycin, which also target the ribosome, ATP release was observed only at high doses of 4-fold and 2.5-fold the MIC, respectively (Figure 9). For puromycin, another ribosome-targeting bacteriostatic drug, very weak leakage was observed at concentrations below the MIC (Figure 9). Because puromycin inhibits the bioluminescence assay at these concentrations, the signal measured at concentrations above the MIC may be underestimated (Figure 5b). Exposure to chloramphenicol at concentrations 2-fold and 4-fold higher than the MIC resulted in significant leakage that appeared linear over time (Figure 9). This is reminiscent of the results we observed with tetracycline and azithromycin. Rifampicin is a transcription initiation inhibitor that induces rapid mRNA decay, resulting in bacteriostatic effects in E. coli. We observed weak ATP leakage with a time lag of approximately 30 minutes. The signal became stronger at concentrations above the MIC (50 μg / mL). Below the MIC, the trace was monophasic (Figure 9). We conclude that bacteriostatic antibiotics, when used at bacteriostatic concentrations, induce very weak ATP efflux, and thus the ATP leakage observed with bactericidal drugs is an important signature of the drug's lethal action (Figure 10).

[0125] Bactericidal antibiotics stimulate ATP release in minimal growth media. We performed ATP release assays in minimal medium using exponentially growing cells.

[0126] ATP release was monitored by MIC values ​​or serial dilutions of antibiotics (Figure 11). For all antibiotics, the intensity of the ATP release signal was weaker in minimal medium compared to rich medium. For example, the values ​​were 10-fold and 3-fold lower for ampicillin and polymyxin, respectively. Thus, the assay demonstrated higher sensitivity in detecting ATP in minimal medium. This result indicated that ATP leakage was less pronounced in minimal medium. We also noted that monophasic traces for polymyxin and cephalexin contrast to the biphasic or multiphasic traces observed for these drugs in rich medium, respectively. This observation also applied to the aminoglycosides, neomycin, gentamicin, and apramycin (Figure 11). The lag time of ATP release remained unchanged or slightly increased, except for ofloxacin, which was reduced by 2-fold (Figure 11). We conclude that the observation of ATP leakage from bactericidal antibiotics in rich media is similar in minimal media.

[0127] ATP leakage assay specifically designed to identify bactericidal compounds It is well known that Streptomyces can produce many secondary metabolites, especially antibiotics. Bacteria of the genus Streptomyces provide approximately 60% of the antibiotics used in modern medicine. The goal of this study was to develop an assay that could be performed with a variety of medically relevant reporter strains, such as pathogens, multidrug-resistant bacteria, or biofilm-forming bacteria. To investigate whether Streptomyces bacteria can produce antibiotics detectable in the assay described herein, we used the neomycin-producing bacterium Streptomyces fradiae and the reporter bacterium Escherichia coli (Figure 19). We also confirmed the feasibility of using a Gram-positive reporter strain, Bacillus luteus (Figure 12). Cultivation of the producer strains was performed in triplicate. The goal was to create a robust assay for testing substantial libraries using only chemical components and culture supernatants from bacteria, cyanobacteria, and fungi. A strong, monophasic ATP leakage signal was measured when culture supernatant from Streptomyces fradiae was added (Figure 13). However, a significant signal was also observed without the addition of reporter bacteria. Similar results were also observed with the supernatants of two mutants of Streptomyces fradiae: a non-producing multimutant (DSM41550) and a single mutant (Δneo6) (data not shown). This result indicated that some components of the supernatant reacted with the luminescent reagent to produce luminescence, and therefore the initial protocol should be optimized.

[0128] Assay optimization Further investigation revealed that the rich medium (LB or TSB) used in the preliminary assay reacted with the supernatant, generating a false-positive signal in the absence of reporter bacteria (Figure 14). When the rich medium LB (or TSB) was replaced with minimal medium MOPS-glucose 0.4%, this false-positive signal disappeared (Figure 15). This suggests that a component in the rich LB medium catalyzed ATP production from precursors or reacted available ATP molecules with the luciferase-luciferin reagent.

[0129] Next, adjustments were made to increase the sensitivity of the test, allowing it to detect the presence of bactericidal components at concentrations below the MIC. The adjusted parameters were: OD 600The volume of reporter cells, the volume of bioluminescence reagents, and the total volume of the assay were also successfully reduced. Because Streptomyces fradiae is a nonmotile bacterium, it produces many extracellular proteases to survive in the environment. Furthermore, given that these proteases are variably produced, we chose to eliminate proteases in the supernatant to eliminate this source of variability and improve sensitivity, because proteases may degrade luciferase. First, we attempted to inhibit the action of proteases with phenylmethanesulfonyl fluoride (PMSF), a serine protease inhibitor. Indeed, Streptomyces fradiae can produce serine proteases. Addition of PMSF did not significantly improve the results (Figure 16). We concluded that other proteases (not limited to serine proteases) and / or other molecules in the supernatant were inhibiting signal acquisition.

[0130] To address the adverse effects of proteases on the measurement of secondary metabolites produced by Streptomyces fradiae, we decided to filter the supernatant using a membrane with a 5,000 Da cutoff. Two key observations should be highlighted when filtering the supernatant (Figure 16). First, we observed a two-fold increase in signal when filtering the supernatant from the neomycin-producing strain. Furthermore, the pattern observed when filtering the supernatant was somewhat reminiscent of that observed with pure neomycin: a strong biphasic signal for the initial ATP release, but with slow kinetics (Figure 16). The observed delay was due to the rich TSB medium (Figure 17). We concluded that filtration removes proteins and large molecules that interfere with assay performance. Furthermore, reducing the assay volume to 9 μL in a 384-well microtiter plate did not result in a loss of sensitivity (Figure 18). By utilizing medium modification, volume reduction, and filtration, the present inventors have succeeded in providing a highly sensitive technique for detecting and identifying the bactericidal component neomycin in the supernatant of Streptomyces fradiae.

[0131] Assays to Detect Variation in Secondary Metabolite Production in Streptomyces fradiae To confirm that the assay described herein is robust enough for high-throughput screening, we tested the supernatants of neomycin-incompetent Streptomyces fradiae mutants. We used strain DSM41550, which contains multiple mutations, including those involved in neomycin production, and strain ΔNeo6, which lacks genes involved in neomycin synthesis. As with the wild-type producer, all mutant Streptomyces fradiae cultures were performed in triplicate, and the supernatants of the two cultures were tested. As expected, the wild-type and mutant strains did not show identical results. The supernatant of the wild-type producer strain generated a strong biphasic signal, as shown in Figure 19c. However, no signal was observed in the supernatants of the non-producing strains, either the multi-mutant strain (DSM41550) or the single-mutant strain (Δneo6) (Figure 19c). This result indicated that the neomycin biosynthetic pathway was inactivated by the mutations. The inventors concluded that by using an optimized assay it is possible to detect bactericidal compounds in culture supernatants of drug-producing bacteria.

[0132] Antibiotic-resistant strains or biofilms as reporter cells There is strong interest in identifying novel families of bactericides that are effective against pathogenic strains that have acquired resistance to currently used drugs. It would also be advantageous to discover drugs that are highly effective against biofilms formed by multidrug-resistant strains. Therefore, we tested whether the assay described herein reports drug-dependent ATP leakage on biofilms. After an initial ATP release by the multidrug-resistant S. aureus USA300 strain (Figure 20a), which was found to be drug-dependent and attributed to physical manipulation, ATP release was induced by the drug neomycin (Figure 20b). This response was also observed when using filtered supernatants from the neomycin-producing Streptomyces fradiae strain, but not unfiltered solutions, further validating the optimized protocol disclosed herein (Figure 20b). Furthermore, consistent with our previous findings, supernatants obtained with the Streptomyces fradiae mutant DSM did not produce a positive response (Figure 20b). These results demonstrate that it is possible to search for novel bactericidal compounds in the supernatants of drug-producing bacteria that may be active against multidrug-resistant strains, even when present in the form of a biofilm (Figure 21).

[0133] Estimation of MIC values ​​(minimum inhibitory concentrations) from bioluminescence data We observed a delay between the moment of antibiotic addition and the detection of the bioluminescence signal reporting ATP (or analog) leakage. This delay shortens as the antibiotic concentration increases. For most antibiotics, the concentration-lag time relationship can be fitted with an exponential decay. In our data, the MIC values ​​were always found from the entry phase to a plateau where the lag time shortens. This plateau represents the limit where the lag time does not change significantly with increasing antibiotic concentration. To illustrate this observation, we subtracted the plateau value of the lag time value for a given antibiotic from the lag time of the known MIC for that drug. This difference was then compared to the total amplitude of the lag time change and expressed as a percentage, denoted X in Figure 22. We found that for the set of antibiotics tested, the value of X was always less than 307% of the total amplitude observed (Figure 22 and Table 3 below).

[0134] [Table 3]

[0135] One possible explanation for this observation is that at this concentration of antibiotic, when a plateau is reached, almost all bacteria are affected by the antibiotic and respond simultaneously. Increasing the antibiotic dose cannot further shorten the delay, since the drug has already acted on almost all bacteria. Therefore, when testing the susceptibility of a strain to an antibiotic, the MIC can be estimated to be in the low concentration range corresponding to segment X. Preferred embodiments of the present invention are as follows. [1] A method for screening compounds for bactericidal activity, comprising: a) providing one or more test bacterial samples comprising live bacteria in a culture medium; b) adding a mixture of luciferin and thermostable luciferase to the test bacterial sample; c) adding a candidate composition to the test bacterial sample; d) incubating the test bacterial sample to which the mixture of luciferin and thermostable luciferase and the candidate composition have been added at 20-60°C (a temperature selected in accordance with the luciferase thermostability parameters), preferably 35-37°C, and measuring bioluminescence in real time; Including, the optical density at 600 nm (OD600) of live bacteria in the test bacterial sample after steps a), b), and c) are performed is at least 0.0002; The method, wherein an increase in bioluminescence measured in the test bacterial sample in step d) indicates that the candidate composition added to the test bacterial sample in step c) contains at least one compound with bactericidal activity. [2] The method according to [1], wherein the live bacteria of the test sample prepared in step a) have not been subjected to mechanical or chemical stress, in particular, the live bacteria of the test sample prepared in step a) have not been subjected to centrifugation or osmotic shock. [3] The method according to [1] or [2], wherein the live bacteria in the test sample are selected from the group consisting of antibiotic-resistant bacteria, including multidrug-resistant bacteria, pathogenic bacteria, planktonic cells, and bacterial cells in biofilms. [4] The live bacteria in the test sample are - Antibiotic-resistant "priority pathogens" of the ESKAPE group - Helicobacter pylori, Campylobacter spp., Salmonellae, Neisseria gonorrhoeae, Streptococcus spp., Haemophilus infulenzae, Shigella spp., coagulase-negative Staphylococci, Mycobacterium tuberculosis, Pseudomonas spp., Enterococcus faecalis, Escherichia coli, Proteus mirabilis, Serratia marcescens and Citrobacter freundii The method according to any one of [1] to [3] above, wherein the compound is selected from the group consisting of: [5] The method according to any one of [1] to [4] above, wherein the culture medium used in step a) is a minimal medium preferably supplemented with a carbon source, more preferably glucose. [6] The method according to any one of [1] to [5], wherein the OD600 of the live bacteria in the test bacterial sample after steps a), b) and c) are performed is between 0.1 and 0.3, preferably between 0.1 and 0.2. [7] The method according to any one of [1] to [6], wherein in step b), the thermostable luciferase is selected from a modified luciferase having GenBank Release 243 Accession No. AAE43251.1 or a modified luciferase having GenBank Release 243 Accession No. AFI79290. [8] The method according to any one of [1] to [7] above, wherein the candidate composition is a purified compound, preferably a chemical compound or a biological compound, such as a phage. [9] The method according to any one of [1] to [7] above, wherein the candidate composition is a complex mixture, preferably a bacterial supernatant or extract. 〔10〕 The method of claim 9, wherein the composite mixture is filtered before being added in step c).

[11] The method is carried out in a microplate, and at the end of step c), the microplate preferably comprises: i) at least one screening well containing a test bacterial sample, a mixture of luciferin and thermostable luciferase, and a candidate composition; ii) at least two negative control wells, one containing a test bacterial sample, a mixture of luciferin and thermostable luciferase, but not containing the candidate composition or the known bactericidal composition, and the other containing culture medium, but not containing the test bacterial sample, the mixture of luciferin and thermostable luciferase, the candidate composition, or the known bactericidal composition; and / or iii) at least one positive control well containing a test bacterial sample, a mixture of luciferin and thermostable luciferase, and a known bactericidal composition or ATP; The method according to any one of [1] to

[10] above, comprising:

[12] A method for determining the susceptibility of a bacterial sample derived from a subject suffering from a bacterial infection to a group of known antibiotics, comprising: a) inoculating a culture medium with a bacterial sample from a subject suffering from a bacterial infection and, optionally, amplifying said bacteria in said sample; b) dividing the bacterial sample of step a) into a number of subsamples, with the number of samples being at least equal to the number of known antibiotics to be tested; c) adding a mixture of luciferin and thermostable luciferase to each subsample; d) spiking one or more subsamples with each of a group of known antibiotics; e) incubating the subsample to which the mixture of luciferin and thermostable luciferase and the known antibiotic have been added at a temperature of 20 to 60°C, preferably 35 to 37°C, and measuring bioluminescence in real time; Including, the optical density at 600 nm (OD600) of the live bacteria in the subsample after steps a), b), c) and d) have been performed is at least 0.0002; The method, wherein an increase in bioluminescence measured in the subsample in step e) indicates that the bacterial sample from the subject is sensitive to the added concentration of the known antibiotic added to the subsample in step d).

[13] In step a), i) the live bacteria of the sample have not been subjected to mechanical or chemical stress, in particular the live bacteria have not been subjected to centrifugation or osmotic shock, and / or ii) The method according to

[12] above, wherein the culture medium is a minimal medium supplemented with glucose.

[14] The method according to

[12] or

[13] , wherein in step b), the thermostable luciferase is selected from a modified luciferase having GenBank Release 243 Accession No. AAE43251.1 or a modified luciferase having GenBank Release 243 Accession No. AFI79290.

[15] A method for evaluating 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 subsamples; c) adding a mixture of luciferin and thermostable luciferase to said subsample; d) adding various concentrations of a bactericidal compound to said sub-samples; e) incubating the sub-sample to which the mixture of luciferin and thermostable luciferase and the bactericidal compound has been added at a temperature of 20-60°C, preferably 35-37°C, and measuring bioluminescence in real time, wherein the optical density at 600 nm (OD600) of the live bacteria in the sub-sample after steps a), b), c) and d) have been performed is at least 0.0002; f) for each concentration of bactericidal compound tested, determining the lag time between the time the bactericidal compound is added and the time an increase in bioluminescence signal is detected; g) expressing lag time as a function of the bactericidal compound concentration; h) constructing an exponential decay curve fitted to the lag time measurement points as a function of the bactericidal compound concentration; i) determining the lowest bactericidal compound concentration at the plateau of the exponential decay fitting curve; j) determining the lag time amplitude of the exponential decay fitting curve; k) assessing the MIC, wherein the MIC is: the antibiotic concentration corresponding to a lag time equal to (lag time at plateau + 0.3 × lag time amplitude) on the exponential decay fitting curve; The lowest bactericidal compound concentration at the plateau of the exponential decay fitting curve and and the step evaluated as being included between A method comprising:

[0136] References TIFF0007795745000006.tif241170 TIFF0007795745000007.tif245170 TIFF0007795745000008.tif177170

Claims

1. 1. A method for screening compounds for bactericidal activity comprising: a) providing one or more test bacterial samples comprising live bacteria in a culture medium; b) adding a mixture of luciferin and thermostable luciferase to the test bacterial sample; c) adding a candidate composition to the test bacterial sample 2 to 15 minutes after step b); d) incubating the test bacterial sample to which the mixture of luciferin and thermostable luciferase and the candidate composition have been added at 20-60°C, preferably 35-37°C, and measuring bioluminescence in real time; Including, the optical density at 600 nm (OD600) of live bacteria in the test bacterial sample after steps a), b) and c) have been performed is at least 0.0002; wherein an increase in bioluminescence measured in the test bacterial sample in step d) indicates that the candidate composition added to the test bacterial sample in step c) contains at least one compound with bactericidal activity.

2. 2. The method of claim 1, wherein the live bacteria of the test sample provided in step a) have not been subjected to mechanical or chemical stress.

3. 3. The method of claim 1 or claim 2, wherein the live bacteria in the test sample are selected from the group consisting of antibiotic-resistant bacteria, including multidrug-resistant bacteria, pathogenic bacteria, planktonic cells, and bacterial cells in biofilms.

4. The live bacteria in the test sample are - ESKAPE Group's antibiotic-resistant "priority pathogens" - Helicobacter pylori, Campylobacter spp., Salmonella spp., Neisseria gonorrhoeae, Streptococcus spp., Haemophilus influenzae, Shigella spp., coagulase-negative Staphylococci, Mycobacterium tuberculosis, Pseudomonas spp., Enterococcus faecalis faecalis, Escherichia coli, Proteus mirabilis, Serratia marcescens, and Citrobacter freundii The method of any one of claims 1 to 3, wherein the compound is selected from the group consisting of:

5. The method according to any one of claims 1 to 4, wherein the culture medium used in step a) is a minimal medium.

6. 6. The method according to any one of claims 1 to 5, wherein the OD600 of live bacteria in the test bacterial sample after steps a), b) and c) have been carried out is comprised between 0.1 and 0.3, preferably between 0.1 and 0.

2.

7. 7. The method of any one of claims 1 to 6, wherein in step b), the thermostable luciferase is selected from the modified luciferase having GenBank Release 243 Accession No. AAE43251.1 or the modified luciferase having GenBank Release 243 Accession No. AFI79290.

8. The method of any one of claims 1 to 7, wherein the candidate composition comprises a purified compound, preferably a chemical or biological compound, such as a phage.

9. The method of any one of claims 1 to 7, wherein the candidate composition is a complex mixture, preferably a bacterial supernatant or extract.

10. 10. The method of claim 9, wherein the composite mixture is filtered before being added in step c).

11. If the method is carried out in a microplate, at the end of step c) the microplate preferably comprises: i) at least one screening well containing a test bacterial sample, a mixture of luciferin and thermostable luciferase, and a candidate composition; ii) at least two negative control wells, one containing a test bacterial sample, a mixture of luciferin and thermostable luciferase, but not containing the candidate composition or the known bactericidal composition, and the other containing culture medium, but not containing the test bacterial sample, the mixture of luciferin and thermostable luciferase, the candidate composition, or the known bactericidal composition; and / or iii) at least one positive control well containing a test bacterial sample, a mixture of luciferin and thermostable luciferase, and a known bactericidal composition or ATP; The method according to any one of claims 1 to 10, comprising:

12. 1. A method for determining the susceptibility of a bacterial sample from a subject suffering from a bacterial infection to a group of known antibiotics, comprising: a) inoculating a culture medium with a bacterial sample from a subject suffering from a bacterial infection and, optionally, amplifying said bacteria in said sample; b) dividing the bacterial sample of step a) into a number of subsamples, with the number of samples being at least equal to the number of known antibiotics to be tested; c) adding a mixture of luciferin and thermostable luciferase to each subsample; d) 2 to 15 minutes after step c), adding each of a group of known antibiotics to one or more subsamples; e) incubating the subsample to which the mixture of luciferin and thermostable luciferase and the known antibiotic have been added at a temperature of 20-60°C, preferably 35-37°C, and measuring bioluminescence in real time; Including, the optical density at 600 nm (OD600) of the live bacteria in the subsample after steps a), b), c) and d) have been performed is at least 0.0002; wherein an increase in bioluminescence measured in the subsample in step e) indicates that the bacterial sample from the subject is sensitive to the added concentration of the known antibiotic added to the subsample in step d).

13. In step a), i) the live bacteria of the sample have not been subjected to mechanical or chemical stress, and / or ii) the culture medium is a minimal medium supplemented with glucose; The method of claim 12.

14. 14. The method of claim 12 or 13, wherein in step b), the thermostable luciferase is selected from the modified luciferase having GenBank Release 243 Accession No. AAE43251.1 or the modified luciferase having GenBank Release 243 Accession No. AFI79290.

15. 1. 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 subsamples; c) adding a mixture of luciferin and thermostable luciferase to the subsample; d) adding various concentrations of a bactericidal compound to said sub-samples; e) incubating the sub-sample to which the mixture of luciferin and thermostable luciferase and the bactericidal compound has been added at a temperature of 20-60°C, preferably 35-37°C, and measuring bioluminescence in real time, wherein the optical density at 600 nm (OD600) of the live bacteria in the sub-sample after steps a), b), c) and d) have been performed is at least 0.0002; f) for each concentration of bactericidal compound tested, determining the lag time between the time the bactericidal compound is added and the time an increase in bioluminescence signal is detected; g) expressing lag time as a function of the bactericidal compound concentration; h) constructing an exponential decay curve fitted to the lag time measurement points as a function of the bactericidal compound concentration; i) determining the lowest bactericidal compound concentration at the plateau of the exponential decay fitting curve; j) determining the lag time amplitude of the exponential decay fitting curve; k) assessing the MIC, wherein the MIC is: the antibiotic concentration corresponding to a lag time on the exponential decay fitting curve equal to (lag time at plateau + 0.3 x lag time amplitude); The lowest bactericidal compound concentration at the plateau of the exponential decay fitting curve and and the step evaluated as being included between A method comprising:

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