FR3046797A1 - METHOD FOR THE RAPID DETECTION OF SENSITIVITY OF DRUG MICROORGANISMS - Google Patents

Abstract

The present invention relates to a method for determining the sensitivity of a microorganism to at least one compound and / or a physical agent. The present invention also relates to the use of the method for rapidly determining the sensitivity of a microorganism to determine the minimum inhibitory concentration of a microorganism to at least one compound, for example an antibiotic, and / or a physical agent. The present invention finds application in particular in the analytical fields, biological research, enzymology, in the pharmaceutical field, in the field of diagnosis and / or in the medical field and / or in the field of disinfection, for example surface .

Description

METHOD FOR THE RAPID DETECTION OF SENSITIVITY OF DRUG MICROORGANISMS

Technical area

The present invention relates to a method for determining the sensitivity of a microorganism to at least one compound and / or a physical agent.

The present invention also relates to the use of the method for rapidly determining the sensitivity of a microorganism to determine the minimum inhibitory concentration of a microorganism to at least one compound, for example an antibiotic, and / or a physical agent.

The present invention finds application in particular in the analytical fields, biological research, enzymology, in the pharmaceutical field, in the field of diagnosis and / or in the medical field and / or in the field of disinfection, for example surface .

In the description below, references in brackets ([]) refer to the list of references at the end of the text.

State of the art

Antibiotic tests are tests to determine the sensitivity of a microorganism to an antibiotic. This determination is generally carried out for pathogenic microorganisms. The aim of the antibiogram is to determine the Minimal Inhibitory Concentrations (MIC) of a bacterial strain against various antibiotics. According to the definition of the World Health Organization (WHO), the MIC is the lowest concentration of antibiotic capable of causing a complete inhibition of the growth of a given bacterium, appreciable to the naked eye, after a period given incubation. The determination of this value is not very precise but it is consecrated by use and it benefits from a large amount of information collected about it, for example as mentioned in the document "General technical conditions. Bulletin of the

French Society of Microbiology, 1993, 8, 156-66 [1], and in the document Clinical Microbiological Infection 1996, 2, Suppl. 1 [2], Following the recommendations of the WHO Expert Committee on Biological Standardization (Recommendations of the WHO Expert Committee on Biological Standardization, Technical Reports No. 610, 1977 [3]) , the French Society of Microbiology has created an Antibiogram Committee (CA-SFM) to determine the critical values that delineate the clinical categories (previously designated as therapeutic categories) and to propose a guide for determining the sensitivity of bacteria antibiotics. The critical values defined for the concentrations and diameters of the zones of inhibition, as well as the specific recommendations for certain species or for certain groups of antibiotics are published in an annual communiqué.

Methods and methods known to perform an antibiogram are known and summarized in particular in the recommendations of the Committee of the Antibiogram of French Society of Microbiology (2010 Recommendations. Committee of the Antibiogram of the French Society of Microbiology [4]).

In particular the CA-SFM protocols for the determination of the MIC in a liquid medium describe the conditions and media for their implementation. According to the "general technical conditions for dilution methods ..." (Antibiogram Committee of the French Society of Microbiology, Recommendations 2010, page 4, [4]) the recommended medium is the medium of Mueller-Flinton, with or without supplementation. In particular, this document describes the preparation of the inoculum: "Inoculum: starting from a culture of 18-24 hours on nonselective agar medium, prepare a suspension in Mueller-Hinton broth or in saline solution (0.9% NaCl) equivalent to the standard McFarland 0.5 (~ 108 CFU / ml). This suspension can also be prepared from a Mueller-Flinton broth culture obtained after incubation at 37 ° C in a shaking water bath for 3 to 5 hours, and the density is adjusted to the standard 0.5 "McFarland standard.

According to the same CA-SFM recommendations, the results are obtained in 18h, 24h up to 72h depending on the growth rate of the bacterial strains. This delay is the consequence of the definition of the MIC (Minimum Inhibitory Concentration): the lowest concentration of antibiotic capable of causing a complete inhibition of the growth of a given bacterium, appreciable to the naked eye, after a period of given incubation. This important delay is a handicap for the therapist.

There is therefore a real need in the state of the art to find a procedure / means for determining more quickly, for example in less than 18 hours, the value of the MIC.

It is common, for a patient presenting clinical signs for which an infection is suspected, during its examination in OJ by a doctor and / or practitioner, for example a Monday and sampled the same day, between 12 and 24 hours, even one night are necessary to isolate the pathogenic bacterium present in said sample, for example isolation on agar plate, Petri, according to the protocol described by CA-SFM. After this isolation phase, an antibiogram may finally be carried out from one or more isolated colonies taken from the surface of said agar. Thus, the time to obtain a result is at least 18 hours or even up to 72 hours. These delays are important, much more important than most deadlines for results in the Laboratory of Medical Analysis.

In addition, to determine the sensitivity of a microorganism to an antibiotic or active principle, the skilled person is encouraged to follow the recommendations of the Committee of the antibiogram of the French Society of Microbiology (CA-SFM), by testing several reason 2 dilutions around the critical concentration value and also providing for one to several control measure (s) without antibiotic or active ingredient. The determination of the MIC according to these recommendations by appreciating the turbidity of the culture medium with the naked eye can be performed after at least 18 hours.

There is therefore a real need to have a method, including an antibiogram simple to implement, fast, allowing early detection of the MIC.

There is also a real need for a method for determining the sensitivity of a microorganism to an antibiotic overcoming the aforementioned problems of the prior art, including a method for obtaining results in a period of less than 18 hours .

In addition, there is a real need for those skilled in the art to have a method for obtaining rapid results, for example available in less than 6 hours with similar results and consistent with those obtained with methods of the invention. art, especially according to the recommendations of the CA-SFM.

In particular, there is a real need to find a method and / or a means to obtain a reliable result within 6 hours, in particular to the extent that this result would allow the therapist and / or doctor to know the result of the antibiogram at most one day after osculation / sampling (at D + 1), for example in the day of Tuesday if the admission / osculation / sampling was carried out on Monday.

This rapid obtaining of the result will allow the doctor / therapist who prescribes a presumptive, probabilistic treatment to be able to adapt / modify the treatment according to the result of the antibiogram: for example if the pathogenic bacterium isolated from the sample is resistant to the disease. antibiotic prescribed and that one day ahead of the usual processes.

There are in the state of the art processes that can accelerate bacterial growth, for example via the use of a particular culture medium more adapted to the bacterial species, increase of the glucose level, adaptation of the rate of oxygen, or carbon dioxide, etc. However, the use of these methods does not allow obtaining a result, including an antibiogram in times less than 18 hours.

There exists in the state of the art a method for determining the sensitivity of a bacterial strain forming a biofilm to an agent such as an antibiotic, or an antifungal in the case of fungi, for example the method described in the international application WO 2005/090944 [5]. However, the process described in this document does not make it possible to determine the sensitivity of a non-biofilm forming microorganism. Indeed, the method described in the international application WO 2005/090944 is based on the measurement of mobility of magnetic microbeads in a culture well (a 96 well microplate well, for example) co-incubated with a biofilm-producing microorganism. When a biofilm has formed, the microbeads are trapped by said biofilm and are no longer mobile, compared to a well in which the biofilm did not form. The method also makes it possible to determine the efficacy of an antibiotic, for example, to inhibit biofilm formation. However, many pathogenic bacteria such as Escherichia coli, Klebsiella sp., Pseudomonas aeruginosa [8] do not spontaneously form biofilm. The method described in this document therefore does not determine the sensitivity of certain pathogenic or non-pathogenic microorganisms and can not therefore be used universally.

There is therefore a real need to find a method to overcome these defects, disadvantages and obstacles of the prior art, in particular a method for controlling the time of obtaining the result of an antibiogram, reduce costs and reduce the time to obtain the antibiogram results.

In particular, there is a real need to find a method and / or device for achieving an antibiogram result in a time less than 7h.

There are also in the state of the art methods using antibodies, for example ELISA tests used for diagnostic purposes.

This method has several disadvantages, for example it requires many controlled washes decreasing its sensitivity. It also includes many stages of manipulation by the skilled person; performing at least two affinity reactions; the mandatory labeling of the second antibody with at least one sensitive and sophisticated marker, the use of a complex device and difficult to set to detect and quantify the signal emitted by the marker. This method is therefore long, expensive, and requires complex instrumentation for its implementation. In addition, the ELISA is used to quantify or semi-quantify an analyte / component supplied into a reaction mixture, usually in a microplate well. This method does not measure revolution of the amount of an analyte in relation to the amount, or its concentration, initial (e).

There are other methods for detecting an affinity reaction using an antibody, for example techniques based on surface plasmon resonance ("plasmon surface resonance"), piezoelectric scales or even a simple observation by microscope in special cases. To do this, the antibody is fixed on a support adapted to the detection technique, the substance is brought into contact for a given time with the antibody fixed on the support, then the reading is carried out directly or after rinsing to eliminate substances which have not reacted by affinity with the antibodies. These methods use in particular diffraction phenomena, especially on gold surfaces, which require very complex devices to obtain the results. In addition, this process requires, in particular, the preparation of the samples under particular pH and / or ionic strength conditions which may give rise to false positive or false negative results.

All of these methods have many disadvantages, for example the instrument necessary to perform the reading is sophisticated and expensive. In addition, they require special supports for fixing said antibodies. Moreover, they require a large number of steps and are dependent on the quality and the amount of antibodies fixed. In addition, they are not suitable for measuring the quantitative evolution of an analyte, in particular a microorganism, in the reaction well.

There is therefore a real need to find a method to overcome these defects, disadvantages and obstacles of the prior art, in particular a method for controlling the time of obtaining the result of an antibiogram, reduce costs and reduce the time to obtain the antibiogram results.

In addition, there is therefore a real need to find a simple method for obtaining antibiogram results, in particular a process that does not require complex devices, or complex steps for preparing samples and / or supports for the implementation and the realization of the antibiogram.

Description of the invention

The present invention solves the problems and disadvantages of the state of the art by providing a method for determining the sensitivity of a microorganism to at least one compound and / or physical agent comprising the following steps: a) introducing into a culture container comprising on the surface of these inner walls a ligand of said microorganism, a microorganism liquid culture medium comprising i. at least two magnetic or magnetizable particles ii. at least one microorganism, and iii. at least one compound and / or a physical agent said at least two magnetic or magnetizable particles resting on a surface immersed by the culture medium in said container, b) determine the sensitivity of the microorganism to said compound and / or physical agent by application of a magnetic field capable of aggregating said at least two magnetic or magnetizable particles, the absence of aggregation of said at least two particles revealing the insensitivity of said microorganism to said compound and / or physical agent.

The inventors are in fact the first to have surprisingly demonstrated that the method according to the invention makes it possible to determine simply and in a time of less than 8 hours the sensitivity of a microorganism to one or more active principle (s). , antibiotic (s) or physical agents.

In particular, the inventors have surprisingly demonstrated that the capture of microorganisms by ligands adsorbed at the bottom of a container makes it possible, depending on the quantity of microorganisms, to immobilize the microparticles.

The inventors have also demonstrated that by incubating microorganisms in the containers, the concentration of microorganisms, the inoculum being lower than the concentration likely to hinder the movement of the microparticles, it is possible to observe a blockage after incubation of the microorganisms in an environment that allows them to grow.

In particular, the generation time, that is to say the doubling, in vitro, of microorganisms, especially those involved in human or animal pathologies, varies for example from 10 minutes for Vibrio cholerae, 20 minutes for Escherichia coli, 20 to 30 minutes for Salmonella typhimurium, 25 to 30 minutes for Staphylococcus aureus, 30 to 40 minutes for Pseudomonas aeruginosa, 2 hours for yeast Saccharomyces cerevisiae, and up to 20 hours for Mycobacterium tuberculosis, (Microbiochemistry and food, Alain Branger, Educagri Editions, 2012 [9]). In the presence of a compound, for example an antibiotic, a biocide, or a physical action microbial growth may slow down, for example partial bacteriostasis, or stop completely. For microorganisms with a doubling time of 40 minutes, an incubation of 2h corresponds to 3 generation times, a multiplication of 23, or a multiplying factor of 8. For a microorganism having an exceptionally long generation time, for example of 120 minutes, 3 generation times correspond to 360 minutes, ie 6 hours.

The inventors have demonstrated that in the case of a microorganism doubling in 40 minutes, starting from an inoculum 8 times smaller than the quantity of microorganisms blocking the particles, blocking of the particles is observed when they are subjected to a magnetic field after 2 hours. incubation. In parallel, the same microorganism was incubated under the same conditions, with different doses / concentrations of compounds, for example antibiotic (s), or any other growth inhibiting agent, the inventors have demonstrated that the container comprising the concentration lower from which the particles are not blocked corresponds to the Minimum Inhibitory Concentration (MIC). Advantageously, the inventors have demonstrated that the method of the invention makes it possible to determine the MIC of a compound vis-à-vis a microorganism, for example an antibiotic vis-à-vis a bacterium, in less than 6 hours. Advantageously, the method according to the invention thus makes it possible to considerably shorten the time to obtain the result compared with the conventional antibiogram, namely a reduction of 3 to 4 times in time, the result of which is linked to a measurement of the growth of a given bacterium, appreciable to the naked eye "according to the definition of CA-SFM is obtained after 18 or 24h.

Thus, advantageously, the method according to the invention makes it possible to determine the sensitivity of a microorganism to an antibiotic or to one or more active principle (s) well before the methods known to those skilled in the art. In addition, the method according to the invention advantageously makes it possible to determine the sensitivity of a microorganism before the person skilled in the art can appreciate with the naked eye a growth of the microorganism in the culture medium.

Advantageously, the method of the invention makes it possible to determine the sensitivity of a microorganism to an antibiotic or to an active ingredient in a time approximately three to four times lower than those of the state of the art, thus going from 18-24. at 4-6 hours for the determination time.

In addition, advantageously, the method of the invention allows the determination of the MIC with the naked eye, for example without a particular reading device. Advantageously, the method according to the invention allows the determination of the MIC with the naked eye which may correspond to the first concentration of compound, progressing in a series of reasons 2, in which it is observed with the naked eye a in motion of said magnetic or magnetizable particles by application of a magnetic or electric or electromagnetic field.

In addition, the inventors have surprisingly demonstrated that the method of the invention makes it possible to obtain a reliable and reproducible result with a similarity similar to that of the usual processes.

In the present invention, the term "microorganism" means any microorganism known to those skilled in the art. It may be, for example, bacteria, fungi, algae and / or protozoa.

It may be for example the bacteria chosen from the group, without being limited thereto, comprising Acetobacter aurantius, Actinobacillus actinomycetemcomitans, Agrobacterium tumefaciens, Azorhizobium caulinodans, Azotobacter vinelandii, Bacillus anthracis, Bacillus brevis, Bacillus cereus, Bacillus fusiformis, Bacillus licheniformis, Bacillus megaterium, Bacillus stearothermophilus, Bacillus subtilis, Bacteroides gingivalis, Bacteroides melaninogenicus, Bartonella henselae, Bartonella quintana, Bordetella bronchiseptica, Bordetella pertussis, Borrelia burgdorferi, Branhamella catarrhalis, Brucella abortus, Brucella melitensis, Brucella suis, Burkholderia mallei, Burkholderia pseudomallei Calymmatobacterium granulomatis, Campylobacter coli, Campylobacter jejuni, Campylobacter pylori, Chlamydia pneumoniae, Chlamydia psittaci, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridi tetani, Clostridium welchii, Corynebacterium diphtheriae, Corynebacterium fusiforme, Coxiella burnetii Ehrlichia chaffeensis, Enterococcus avium, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus galllinarum, Enterococcus maloratus, Escherichia coli Francisella tularensis, Fusobacterium nucleatum Gardnerella vaginalis Haemophilus ducreyi, Haemophilus influenzae, Haemophilus parainfluenza, Haemophilus pertussis, Haemophilus vaginalis, Helicobacter pylori, Klebsiella pneumoniae, Klebsiella oxytoca Klebseilla rhinoscleromatis- Lactobacillus acidophilus, Lactobacillus casei, Lactococcus lactis, Legionella pneumophila, Methanobacterium extroquens multiforme Microbacterium, Micrococcus luteus, Mycobacterium avium, Mycobacterium bovis, Mycobacterium diphtheriae, Mycobacterium Intracellulare, Mycobacterium leprae, Mycobacterium iepraemurium, Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium tuberculosis, Mycoplasma fermentans, Mycoplasma genitalium, Mycoplasma hominis, Mycoplasma pneumoniae Neisseria gonorrhoeae, Neisseria meningitidis, Nocardia asteroids Pasteurella multocida, Pasteurella tularensis, Porphyromonas gingivalis, Pseudomonas aeruginosa, Pseudomonas maltophilia, Rhizobium radiobacter, Rickettsia prowazekii, Rickettsia mooseri, Rickettsia psittaci, Rickettsia quintana, Rickettsia rickettsii, Rickettsia trachomae, Rochalimaea henselae, Rochalimaea quintana, Rothia dentocariosa, Salmonella enteritidis, Salmonella typhi, Salmonella typhimurium, Serratia marcescens, Shigella dysenteriae, Streptococcus aureus Streptococcus gallinarum, Streptococcus lactis, Streptococcus mitior, Streptococcus mitis, Streptococcus mutans, Streptococcus oralis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus rattus, Streptococcus saliva Streptococcus sanguis, Streptococcus sobrinus, Treponema pallidum, Vibrio cholerae, Vibrio comma, Vibrio parahemolyticus, Vibrio vulnificus, Xanthomonas maltophilia Yersinia enterocolitica, Yersinia pestis and Yersinia pseudotuberculosis, etc.

It can be any fungus known to those skilled in the art, for example pathogenic or non-pathogenic fungi, for example fungi responsible for pathologies, for example in human health or not, environmental fungi, fungi chosen from group comprising yeasts, for exampleCandida and / or Cryptococcus, for example yeasts responsible for pathologies, for example in human health, for example candidiasis and / or cryptococcosis, and / or the group comprising molds, for example Aspergillus, by example of mold responsible for pathology, for example in human health, for example aspergillosis and / or pulmonary mycosis.

According to the invention, in the case of the use of anaerobic bacteria, the anaerobic culture conditions of the microorganism may be obtained by closing the open end of the culture reactor, for example by means of parafilm, a plug, etc. or by placing said reactor under conditions allowing the development of anaerobic bacteria. It may be, for example, anaerobiosis jars, pouches generating an oxygen depleted atmosphere and enriched with carbon dioxide, anaerobic enclosures with a controlled atmosphere and / or any means known to those skilled in the art.

According to the invention "culture container" means any culture container known to those skilled in the art. It may be for example a culture reactor, wells, tubes or wells for example microdilution plates.

According to the invention the culture container may be for example an enclosure with a closed end, of the tube type, well, etc. or an enclosure having two openings. It may also be a container comprising a closed end so as to form a flat bottom, a container with a closed end so as to form a hemispherical bottom, a container comprising two open ends. When the container comprises two open ends, said container may be configured to allow flow of the culture medium in a constant flow or in a discontinuous flow.

It may be for example microdilution plate type of plate defined for example by the American National Standards Institute and the Society for Biomolecular Screening ("microplates") carrying 96 wells, 384 and even 1536, or conversely 48 or 24 wells or any other number of wells.

It may be for example a culture container consisting of any material known to those skilled in the art. It may be for example plastic, for example polycarbonate, polypropylene, polystyrene, etc., glass, metal.

It may be, for example microplates polycarbonate or polypropylene flat-bottom, conical or round. Advantageously, when the container is made of polycarbonate or polypropylene, this makes it possible to prevent unwanted adhesion of the microorganism to the internal surfaces of the container.

It may be for example polystyrene container, for example any polystyrene known to those skilled in the art. Advantageously, when the internal surfaces of the container are made of polystyrene, the fixing of the ligand of said microorganism can be improved. The person skilled in the art, on the basis of his general knowledge, will be able to adapt / choose the polystyrene suitable for a better fixation of the ligand, for example as a function of these physicochemical properties, for example hydrophilic or hydrophobic.

According to the invention, the culture medium may be any culture medium known to those skilled in the art and / or commercially available in which at least one microorganism is likely to develop. It may be for example a natural or synthetic environment. This may be, for example, a culture medium for bacterial growth, for example BHI (Brain Heart Infusion) medium, LB medium (Lysogeny broth), MH medium ( medium Mueller-Hinton "), glucose broth, yeast culture medium, for example Sabouraudr medium The skilled person share this general knowledge will choose the culture medium adapted to the microorganism.

According to the invention, the term "ligand of the microorganism" is understood to mean any ligand known to those skilled in the art. It may be for example a ligand chosen from the group comprising antibodies, peptides, aptamers.

In the present invention, the antibodies can be any antibody known to those skilled in the art and / or commercially available. It may be, for example, monoclonal antibodies, polyclonal antibodies or a mixture thereof. These may be, for example, commercially available antibodies, for example antibodies marketed by AbCam, Acris, Fitzgerald, GeneTex or ThermoFisher Scientific.

In the present invention, the peptides may be any peptide known to those skilled in the art and / or commercially available. It may be for example peptides marketed by the company Clinisciences, Proteomic Solutions, Biomatik, Casio, Genscript.

In the present, aptamers can be any aptamer known to those skilled in the art and / or commercially available. It may be for example aptamers marketed by the company Aptagen, IBA, Novaptech.

Herein, the "microorganism ligand" may be a specific or non-specific ligand of the microorganism.

According to the invention, the nonspecific ligand may be any ligand known to those skilled in the art and / or commercially available. It may be for example a ligand chosen from the group comprising Protein A, Protein G and Protein L.

According to the invention, the ligand of the microorganism may be attached to the surface of the inner walls of the container at a concentration of from 0.1 to 20 μg / ml, for example from 0.5 to 5 μg / ml, for example equal to 6 μg / ml. / ml.

According to the invention, the ligand of the microorganism may be attached to the surface of the inner walls of the container by any method and / or means known to those skilled in the art. It may be for example a passive adsorption, commonly called "coating", for example comprising incubating the ligand in the culture container for adsorbing said ligand on the walls of said container and then washing the container in order to eliminate unbound ligands. It may be for example a process using coupling polymers, for example polypropylene or polycarbonate, for example associated with silylsulfonyl azides.

According to the invention, the surface of the inner walls of the culture container may comprise one or more ligand (s) attached to their surface. When the surface of the inner walls of the culture container comprises a plurality of ligands attached to the surface, the method of attachment may comprise a first ligand binding step, for example a ligand selected from the group consisting of Protein A, Protein G, Protein L followed by a second binding step, for example ligand selected from the group comprising antibodies.

According to the invention, said at least two particles may be selected from the group consisting of an electrically charged particle, a magnetic particle, a particle coated with at least one magnetic layer, a magnetizable particle, a particle coated with a magnetizable layer, a mixture of two or more of these particles. In fact, it may be any particle for implementing the present invention.

According to the invention, the particles rest on a surface immersed in the culture medium. Said particles are in stable position, that is to say at rest, in the absence of the magnetic field, or electric, or electromagnetic. Advantageously, said particles may be particles of any form suitable for the implementation of the present invention, for example in the form of a ball, a puck, an asymmetric geometric shape, for example with a flat face, etc.

Any suitable size of magnetic or magnetizable particles may be used. The size may be chosen for example depending on the size of the microorganism, and / or the size of the medium containing the culture medium for carrying out the method of the invention and / or the microorganism.

For example, the magnetic or magnetizable particle may have a size of, for example from 10 nm to 100 μm, for example from 0.1 to 10 μm.

According to the invention, the particles may be of identical or different sizes, the proportion between the particles of different sizes, relative to the microorganism in question, which can modify the sensitivity of the test.

When the particles are of different sizes, the small particles may have a size, for example, from 10 nm to 1 μm, for example from 100 to 500 nm, and the large particles may have a size, for example, from 1 μm to 100 μm. for example from 1pm to 10pm, for example from 1pm to 5pm. Of course, the respective size of the large and small particles is chosen so as to determine different stages of development.

According to the invention, it is also possible to use a plurality of particles of identical sizes with different magnetization. The movement of the particles will be dependent on their magnetization, the blocking being kept constant because of the relative size of the particles due to their size.

According to the invention, the process of the invention can be implemented with a plurality of particles, for example from 2 to 10,000,000, from 1,000 to 10,000,000, from 1,000 to 1,000,000, from 10,000 to 10,000,000. 1,000,000, 100,000 to 1,000,000, 10,000 to 100,000, preferably 1,000 to 1,000,000 particles.

According to the invention, the particles can be illuminated, for example by means of a light source. The illumination advantageously makes it possible to increase the contrast between the particles and the solution.

According to the invention, said at least two particles are preferably particles generating a detectable signal. The detection of this signal will depend on the properties of the particles. By way of example, they may be fluorescent, FRET fluorescent, phosphorescent, radioactive, chemiluminescent, reflective or colored particles.

For example, in the case where the at least two particles are fluorescent, the fluorescence emitted by the particles can be detected, for example visually, and / or by any optical means known to those skilled in the art. Said at least two particles may for example be illuminated to follow its movement by means of a light source, for example by a laser beam.

For example, in the case of using FRET technology (Fluorescence Resonance Energy Transfer), the fluorescence emitted by a first particle excited by a wavelength will itself excite the second molecule that will generally emit fluorescence. a wavelength very far from the first exciting wavelength, and after a time such that the background noise emission due to the first excitation is extinguished. This transfer of the first type of particle to the second can only be done if the particles are very close and is proportional to the number of particles collected / aggregated.

For example, in the case where the at least two particles are phosphorescent, these particles can be visualized, for example visually, by any optical means known to those skilled in the art.

For example, in the case where the at least two particles are radioactive, these particles can be detected even through optically opaque liquids or culture media, as well as through optically opaque microdilution plates by any detection device. radioactivity emitted known to those skilled in the art, especially the conventional method of revelation on autoradiographic film. It is then sufficient to press the sensitive film under the microdilution plate and then reveal the bottom image of said microdilution plate.

For example, in the case where the at least two particles are chemiluminescent, the detection of the particles can be formed by adding to the medium the chemical reagent allowing the emission of light energy by the particles. The detection of this signal may be for example visual and / or by any means known to those skilled in the art, in particular by the use of a CCD camera ("Charge Coupled Device") sensitive to wavelengths emitted, which sweep ( scan) the wells, the wells of the microdilution plate.

For example, in the case where the at least two particles are reflective, the detection of the particles may be, for example, visual or by any optical means known to those skilled in the art. Advantageously, said at least two particles may for example be illuminated, to follow their movements by means of a light source, for example by a laser beam.

For example, in the case where the at least two particles are colored, the detection of the particle may for example be visual and / or by any means known to those skilled in the art for the detection of colored particles. Advantageously, if the particles are attracted towards the center and bottom of the wells as described in WO 2005/090944, an image can be acquired by a scanner-type apparatus and then analyzed by an image analysis software allowing quantification of the particles. collected / aggregated at the center of the well.

According to the invention, the particles may advantageously be chosen from different colors depending on their size and / or according to their magnetic power.

It may also be an excitable particle, for example a FRET fluorescent particle. Those skilled in the art on the basis of this general knowledge, will adapt / choose the visual properties of said at least two, particle can according to the medium. Advantageously, the detection of the movement of said at least two particles is all the easier as the contrast between said at least two particles and the culture medium is great.

According to the invention, the observation of can be carried out by any means known to those skilled in the art. It may be for example an optical device, for example a microscope, a camera, a document scanner, for example a perfection scanner V600 EPSON, a visual observation.

According to the invention, the observation can make it possible to measure, for example the intensity, the contrast, the image variance, for example via any means known to those skilled in the art, for example an imaging software, for example ImageJ allowing, for example to measure, for example differences in contrast, intensity, for example corresponding to the particles, in areas of an image to another and thus to determine the differences from one observation to another .

According to the invention, the use of signal-emitting particles, for example colored, fluorescent, phosphorescent, luminescent or radioactive particles, may allow, for example, automated observation.

In the present invention, the field for moving said at least two magnetizable particles can be a magnetic field, or an electric field, or an electromagnetic field. Said magnetic field, or electric or electromagnetic field may be generated, for example by a magnet or a solenoid. The magnet may be for example in the form of a bar, tip, piece, etc. or any form suitable for carrying out the present invention. The field may, for example, be applied by any means known to those skilled in the art, for example by pulse, by gradual increase of the electric or electromagnetic field, by electric or electromagnetic field variations or by a combination of these applications.

A progressive increase of the magnetic field, or electric or electromagnetic field can be obtained, for example, by bringing a magnet or a solenoid in a rectilinear or sinusoidal trajectory, or in an oscillating movement with or without an amplitude of oscillation and / or a variable frequency. More complex field variations can be obtained, for example by rotation or by combinations of motions of a magnetized material in proximity to said at least two particles.

Thus, according to the invention, said magnetic, or electrical, or electromagnetic field may be generated by field generating means which may or may not be in motion.

When several particles must be set in motion, the magnetic, or electrical, or electromagnetic field must be able to group said particles on said immersed surface in the culture medium or on said surface of said culture medium.

According to the invention, by compound is meant any compound, for example natural or obtained by synthesis or chemical hemi-synthesis, known to those skilled in the art likely to modify the growth / development of microorganisms. It may be for example a biocide, for example a disinfectant, a protection product, a product for the control of so-called harmful and other species and / or any biocide included in the list according to the directive 98/8 / EC or Regulation (EU) No 528/2012, an antibiotic, antifungal agent.

This may be, for example, an antibiotic chosen from the group comprising fusidic acid, aminoglycosides, beta-lactams, penicillins, beta-lactamase inhibitors, cephalosporins, carbapenems, monobactams, cycloserine. , daptomycin, fosfomycin, lincosamides, macrolides and ketolides, mupirocine, nitroimidazoles, nitrofurans, oligosaccharides, oxazolidinones, polypeptides: bacitracin, glycopeptides, polymyxins and colistin, quinolones, rifamycins, sulfonamides and diaminopyridines, synergistines, tetracyclines, antifungals, or any mixture thereof.

According to the invention, the antibiotic may be in solution in the culture medium, or in dehydrated or freeze-dried form, and / or fixed on the surface immersed in said medium.

The usable binding means of the antibiotic are those known to those skilled in the art for the attachment of an antibiotic to a surface. These may be for example those commercially available, for example marketed by the company NUNC (Denmark), Corning (United States), and Greiner Bio-One (Austria).

According to the invention, one or more compounds may be introduced into the culture medium, for example one or more antibiotics.

Advantageously, when the method according to the invention makes it possible to test different antibiotics alone and / or to look for an effect of combination or combination of antibiotics in a format commonly called "chessboard" (or checkerboard in English). The implementation of the method of the invention with a mixture of antibiotics (combination, combination) may advantageously make it possible to detect possible additive, synergistic and / or antagonistic effects.

By "additive effect" is meant the addition of the actions of at least two antibiotics allowing, for example, to find a combination of antibiotics having the same antibiotic effect as an antibiotic alone, but with lower concentrations.

By "synergistic effect" is meant an improvement in the efficacy of an antibiotic by addition of at least a second antibiotic, this mixture of antibiotics having a greater effect than the addition of the effect of each antibiotic alone .

Finally "antagonistic effect" means the inhibition of the action of an antibiotic on the microorganism by the addition of at least a second antibiotic. This embodiment can make it possible to select combinations, associations of antibiotics ("co-drug") relevant for, for example, inhibit the development of the microorganism.

According to the invention, the antibiotics may also be introduced successively into the medium in order to identify additive, synergistic or antagonistic effects as described above.

Advantageously, the method according to the invention makes it possible to determine / detect and / or identify the sensitivity of microorganisms to compounds. Also, the method according to the invention advantageously makes it possible to identify a new biocide, for example a new antibiotic and / or antifungal. In addition, the method according to the invention may advantageously make it possible to determine the specificity of a compound, for example if it is an antibiotic and / or an antifungal.

According to the invention, the term "physical agent" means temperature, thermal radiation, pressure, radiation or radiation. It may be for example any radiation known to those skilled in the art, for example corpuscular radiation, ripple radiation and / or electromagnetic radiation.

According to the invention, the process may comprise prior to step a) the following steps: A1) introduction into a culture container (a2) comprising a liquid culture medium suitable for the development of said microorganism, i. at least two magnetic or magnetizable particles ii. at least one microorganism for seeding said medium, and iii. at least one compound and / or physical agent A2) keeping the seeded culture medium and said at least two molecules under conditions allowing a development and / or growth of said microorganism, the medium obtained being the medium introduced into the container of the step at).

According to the invention, the container (a2) and the container of step a) may be identical or different. For example the container (a2) may be for example plastic, for example polycarbonate, polypropylene, polystyrene, glass, metal. Advantageously, the container may be a microplate well made of polycarbonate and / or polypropylene with a flat, conical or round bottom. Advantageously, the polycarbonate or polypropylene reduces the attachment of microorganisms to the surface of the inner walls of the container favoring its growth advantageously.

According to the invention, when the process comprises steps A1 and A2, the culture container of step a) may advantageously be different from the container a2. It may be for example a polystyrene container as described above.

According to the invention, the maintenance of the seeded culture medium under conditions allowing a development and / or growth of said microorganism can be carried out for 30 minutes to 7 hours, for example from 1 to 6 hours, from 2 to 5 hours. Advantageously, the culture medium is maintained under conditions allowing growth and / or growth of said microorganism for 2 to 5 hours. Those skilled in the art, by this general knowledge will be able to choose the conditions of pH, temperature, oxygenation, the supply of metabolizable sources and essential components (such as minerals) for development and / or growth of said microorganism.

According to the invention, the process may be carried out in a plurality of containers comprising on the surface of their internal walls a ligand of said microorganism each comprising: said culture medium, said at least two particles, and said microorganism, said compound and or physical agent, said containers comprising a different concentration of said compound and / or physical agent and / or a compound and / or physical agent different from one container to another.

According to the invention, the different containers may be grouped together on the same support, for example on a plate comprising from 1 to 384 wells, for example 6, 12, 24, 48, 96, etc.

According to the invention, the method may advantageously make it possible to determine the Minimum Inhibitory Concentration (MIC) of the planktonic growth, that is to say in the liquid phase, in the culture medium, of said microorganism.

According to the invention, the determination of the minimum concentration of the antibiotic inhibiting the growth of said microorganism, that is to say the Minimum Inhibitory Concentration (MIC) can be achieved by successively or simultaneously implementing the method of the invention in identical conditions using a liquid culture medium suitable for the development of a microorganism, at least two magnetic or magnetizable particles, at least one microorganism and at least one compound in a concentration different from one embodiment to another. Said antibiotic concentration can be established for example according to a geometric sequence of reason Vz from an initial concentration X, for example X, X / 2, X / 4, X / 8, etc., or of any other suite geometrically appropriate. In carrying out the process of the invention, and among the embodiments, the first concentration, that is to say the lowest concentration from which the particles are mobile, reflecting the activity of the antibiotic on the growth of bacteria, corresponds to the minimum inhibitory concentration (MIC).

This estimation of the mobility of the particles can be made simply visually, for example as for tube antibiograms, or advantageously following the analysis of the image by a suitable software, such as for example the BFC Eléments® software developed by the company. BioFilm Control. Thus, the method according to the invention advantageously makes it possible to determine the minimum concentration of antibiotic from which the microorganism is sensitive to the antibiotic in its growth, that is to say the minimum concentration for which the, at least two particles may be set in motion / aggregate by applying the magnetic field in one of said containers.

The method of the invention may also be implemented with a zero compound concentration, said embodiment constituting a growth control of the microorganism in the culture medium. Another control may consist in validating the sterility of the medium in the presence of particles during the test period. Another witness may be to validate that the antibiotic does not cause artifacts in the medium in the presence of particles.

Whatever the mode of implementation of the invention, the method of the invention can advantageously be carried out simultaneously in a plurality of containers.

When the method of the invention is implemented in a plurality of containers, for the movement of the particles in said containers, a plurality of magnetic fields, for example magnets, can advantageously be used. The magnets may for example be fixed on a support so as to allow a juxtaposition of each container in which the method of the invention is carried out with a magnet of the support. The application of the magnetic field on said containers is independent of one container to another. The magnetic field applied to said containers may be identical or different from one container to another, preferably identical.

The method of the invention therefore also advantageously makes it possible to determine the minimum inhibitory concentration of an antibiotic for bacterial growth for said microorganism by carrying out the process of the invention in the different containers in a single step on a single support. .

Advantageously, the method of the invention allows for example to follow the recommendations of the Committee of the Antibiogram of the French Society of Microbiology, relating to the general technical conditions for the dilution methods.

According to the invention, it is also possible to determine, advantageously, the minimum inhibitory concentration of said antibiotic with respect to the growth of said microorganism suspended in each container.

The method of the invention may, for example, be used for the implementation of an antibiogram of assistance with the prescription of the antibiotic (or antibiotics) effective (s) on a microorganism taken from a patient, by application of the method of the invention. This microorganism can then be tested with different antibiotics, for example on a single analysis support, to optimize the treatment of this patient. Application of the method to said microorganism makes it possible to verify that the microorganism is indeed sensitive to antibiotics at a concentration, for example equal to the MIC.

The method of the invention may also make it possible to follow in-vitro the evolution of the MIC of a microorganism of a patient with respect to an antibiotic. This aspect is important for long-term treatments, or for immunocompromised patients prone to chronic infections with the same microorganism. The method of the invention makes it possible to monitor the appearance of resistance to a treatment. The method then makes it possible to find other antibiotics more able to inhibit the growth of said microorganism.

Advantageously, the process of the invention, by the application of the field and the aggregation or not of the particles makes it possible to obtain a rapid and visual result of the sensitivity or not of the microorganism to the compound and / or physical agent. Other advantages may still appear to those skilled in the art on reading the examples below, illustrated by the appended figures, given for illustrative purposes.

Brief description of the figures

FIG. 1 shows that the blocking of the microbeads is proportional to the number of bacteria in the wells and makes it possible to determine the threshold at which the microbeads are blocked.

Figure 2 shows an example of determination of Minimal Inhibitory Concentration (MIC) of 3 antibiotics on strains of Staphylococcus aureus.

Fig. 3 shows photographs corresponding to an example of determination of sensitivity or resistance of microorganisms to a compound as a function of the absence or presence of ligand on the inner surface of the wall of the culture container.

EXAMPLES

Example 1: Determination of the amount of bacteria needed to block the microbeads.

The results are shown in Figure 1.

Several colonies (about 6) of Staphylococcus aureus were taken on a Petri dish containing Mueller-Hinton agar medium and resuspended in liquid Mueller-Hinton medium. The optical density (at 600nm) was measured, considering that a unit of OD corresponds to 1x109 bacteria / ml. A polystyrene plate was previously coated ("coated") with anti-staphylococcus aureus antibodies (Aut \ -Staphylococcus aureus rabbit, purified IgG fraction, Acris). To do this, the plate was incubated overnight at 4 ° C with 200 μl / well with an antibody solution at concentrations 1; 2; 4; 6 or 8 μg / ml in phosphate buffered saline (PBS). The plate was washed 2 times with the plate washer with PBS supplemented with 0.1% Tween-20 to remove the non-adsorbed antibodies on the polystyrene. After the last wash, the wells were left empty. 200 μl of bacteria solution at a concentration of 0 to 1 × 10 8 / ml in Mueller-Hinton medium, in the presence of magnetic microbeads, of the order of 5 × 10 5 to 2 × 10 7 per well, were added and left for 30 minutes to allow capture. bacteria by antibodies and decantation of magnetic microbeads. The microplate was then covered with 10ΟμΙ of contrast liquid and placed 1 minute on a block carrying 96 mini-magnets arranged so that they are centered relative to the wells. The microplate was then placed on a scanner, driven by the BFCE 3 software and an image was captured. The image is then analyzed and a BFI (BioFilm Index) value as described in Chavant et al. [10] was calculated. A value of 0 corresponds to a total blocking of the microbeads whereas a value of 20, or near, corresponds to a total mobility of the balls. All intermediate values are possible.

The plate plan has been defined as follows:

FIG. 1A corresponds to the raw image obtained

Figure 1B corresponds to the graphical representation of the

condition of covering the internal surfaces of the anti-Staphylococcus aureus antibody container at 6 μg / ml, for 2 replicate conditions (1 and 2). As shown in this figure, the greater the number of bacteria present in the medium, the lower the mobility of the particles. In other words, the blockage of the beads is proportional to the number of bacteria present in the medium.

Figure 2

Example 2: antibiograms according to the method of the invention and comparison with the methods of the state of the art.

The determination of the sensitivity or the resistance of the strains, on the basis of their MIC, with different antibiotics was established according to the standard protocol of the Committee of the Antibiogram of the French Society of Microbiology (CA-SFM), in medium Mueller -Flinton liquid dilution reason 2. This protocol is well known to those skilled in the art and is described in the General Technical Conditions for dilution methods (Bulletin of the French Society of Microbiology, 1993, 8, 156- 66 [1] Clinical Microbiological Infection 1996, 2, Suppl.1 [2]). The same values were obtained in liquid BHI medium.

The Sensible (S) or Resistant (R) qualification of a strain is based on the relative position of the MIC determined with respect to the "breakpoint", a value taking into account different parameters. Thus, for a given type of bacterium, and a given antibiotic, a breakpoint value has been established by committees. This breakpoint value serves as a threshold: if the MIC of the strain tested is lower than the breakpoint,

the bacterium is called Sensitive, that is to say a "low" dose of antibiotic is enough to inhibit its growth, if on the contrary the MIC of the strain is greater than the "breakpoint", the strain is Resistant. For some antibiotics, this value is not unique but can spread over several concentrations.

In this example, 2 S. aureus strains were chosen because they had opposite Sensitivity / Resistance profiles for the 3 selected antibiotics.

The strain A929 is fosfomycin (S), erythromycin (S) and rifampicin (R).

Strain B198 is fosfomycin (R), erythromycin (R) and rifampicin (S).

Antibiotics (erythromycin, reference E1300000, fosfomycin, reference F0400000, rifampicin, reference R0700000) came from EDQM (European Directorate for the Quality of Medicines and Healthcare, Council of Europe) and meet the criteria of the European Pharmacopoeia.

The bacteria were resuspended in a tube containing 2 ml of liquid Mueller-Hinton medium and left to grow with light agitation for 2 hours. This step aims to obtain bacteria exponential growth phase and standardize their growth during the 2 hours microplate, in the presence of antibiotics. This growth, in the presence of antibiotics was carried out in polypropylene microplates, with a round bottom, brand and reference Nunc 054309, this type of support being preferred to limit the adhesion of microorganisms at this stage.

Flat-bottom polystyrene microplates brand and reference Costar 3370 were coated with polyclonal rabbit antibodies, anti-staphylococcus aureus, according to the following method. A polystyrene plate was previously coated ("coated") with anti-staphylococcus aureus antibodies (anti-Staphylococcus aureus rabbit, purified IgG fraction, Acris). To do this, the plate was incubated overnight at 4 ° C. with 200 μl / well of a solution of antibody at a concentration of 6 μg / ml in phosphate buffered saline (PBS). The plate was washed 2 times with the plate washer with PBS supplemented with 0.1% Tween-20 to remove the non-adsorbed antibodies on the polystyrene. After the last wash, the wells were left empty.

A second plate, uncoated, served as a control.

In other polypropylene microplates, round bottom, brand and reference Nunc 054309, for each of the conditions tested, 40 .mu.l of antibiotic solution at a concentration 10X was deposited. The deposited solutions were 10 times more concentrated than the desired final concentration because the addition of 360 μΙ of medium containing the bacteria has the effect of diluting them by a factor of 10 (400/40). The concentration expressed in the results is the final concentration, in the presence of bacteria.

Following the addition of the 40 μΙ of antibiotics, 360 μΙ of Mueller-Hinton medium, containing the microbeads at a concentration of about 5 × 10 5 to 2 × 10 7 per well, were added. The starting inoculum of staphylococci corresponds to an OD measurement at 600 nm of 0.04, ie of the order of 4 × 10 7 bacteria / ml. Incubation at 37 ° C takes place for 2 hours.

Following the incubation for 2h in the Nunc 054309 microplate in the presence of antibiotics, 200 μl of the reaction medium of each well (Mueller Hinton medium, bacteria, antibiotics, magnetic microbeads) were transferred to the wells coated with the Costa 3370 microplate ( and uncoated, in control) and left for 30 minutes to allow antibody capture of the bacteria and decantation of the magnetic microbeads.

The microplates were then covered with 100μΙ of contrast liquid and placed 1 minute on a block carrying 96 mini-magnets arranged so that they are centered relative to the wells. The microplates were then placed on a scanner, driven by the BFCE 3 software and an image was captured.

The letter S defines the strain as Sensible (CMI lower than the breakpoint EUCAST) while the letter R defines the strain as Resistant (CMI superior to the breakpoint EUCAST).

The plate scheme is as follows:

The values in the table are in pg / ml (or mg / l).

The value of the breakpoint is indicated in bold in square brackets. From this plate scheme, two plates A and B were established. Plate A was coated with anti-S antibodies. aureus, ligand of the microorganism, whereas the plate B was not. Thus, a plate included culture containers comprising on the surface of their internal walls a ligand of the microorganism (plate A) or not (plate B).

The results obtained after applying the magnetic field are shown in FIG. 3A and 3B.

Figure 3

As shown in FIG. 3B, in the absence of ligand on the inner surface of the wall of the culture container (control condition), no blocking could be observed and a grouping / aggregation of the particles was observed whatever the incubation conditions of the microorganism. Also, it is clear that the ligand attached to the surface of the inner walls of the container is necessary for the determination of the sensitivity or resistance of the microorganism.

In FIG. 3A, it appears that in column 1 (strain S) the microbeads are not blocked from the line C, hence above the value of the breakpoint, whereas in column 2 (strain R) the

Microbeads are mobile only in line A, at a higher concentration than breakpoint.

Similarly, in column 3 (strain S), the microbeads are mobile from line F, while in column 4 (strain R), the microbeads are blocked at all concentrations of erythromycin.

In column 5 (strain R), the microbeads are blocked at all concentrations of rifampicin, while in column 6 (strain S), the microbeads are mobile from line G.

The results obtained are therefore consistent and identical to those obtained with the methods of the state of the art, in particular the reference methods for determining an antibiogram.

In addition, as demonstrated in this example, the method according to the invention advantageously makes it possible to obtain a reliable, consistent result, without implementation of a complex device. In addition, the method according to the invention makes it possible to determine the sensitivity of a microorganism to a compound and to determine its MIC in a time 3 to 4 times less than the known method, for example between 3 and 5 hours.

The method according to the invention, by rapidly obtaining the sensitivity or the resistance of the microorganism to a molecule, for example an antibiotic, to quickly adapt and / or confirm the antibiotic treatment of a patient thereby providing a real clinical advantage, for example by gaining 24 hours in treatment and its possible adaptation.

List of references 1. General technical conditions. Bulletin of the French Society of Microbiology, 1993, 8, 156-66; 2. Clinical Microbiological Infection 1996, 2, Suppl. 1; 3. Recommendations of the WHO Expert Committee on Biological Standardization. Technical Reports No. 610,1977; 4. Recommendations 2010. Antibiogram Committee of the French Society of Microbiology; 5. WO 2005/090944 "Method and apparatus for detecting the formation and development of biofilms in a culture medium" 6. Romling U and Balsalobre C. Biofilm infections, their resilience to therapy and innovative treatment strategies. J Intern Med 2012, 272 (6): 541-561 7. Research on Microbial Biofilms, NIH Parent Grant Announcement. 2002. NIH website. Available: http://grants.nih.gov/grants/guide/pa-files/PA-03-047.html. Accessed 2014 Sept 2.NIH 2002. 8. Olivares E, Badel-Berchoux S, Provot C, Jaulhac B, Prevost G, Bernardi T, Jehl F. The Biofilm Ring Test®: a rapid method for the routine analysis of P. aeruginosa biofilm training kinetics. J Clin Microbiol.

2015 Dec 30. Pci: JCM.02938-15. [Epub ahead of print] 9. Microbiochemistry and food, Alain Branger, Educagri Editions, 2012 10. Chavant P, Gaillard-Martinie B, Talon R, Hebraud M, Bernardi TA new device for rapid evaluation of biofilm formation potential by bacteria .. J Microbiol Methods. 2007; 68 (3): 605-12

Claims (17)

A method for determining the sensitivity of a microorganism to at least one compound and / or physical agent comprising the following steps: a) introducing into a culture container comprising on the surface of these internal walls a ligand of said microorganism, a medium microorganism liquid culture comprising i. at least two magnetic or magnetizable particles ii. at least one microorganism, and iii. at least one compound and / or physical agent said at least two magnetic or magnetizable particles resting on a surface immersed by the culture medium in said container, b) determine the sensitivity of the microorganism to said compound and / or physical agent by application of a magnetic field capable of aggregating said at least two magnetic or magnetizable particles, the absence of aggregation of said particles revealing the insensitivity of said microorganism to said compound and / or physical agent. 2. Method according to claim 1, said method comprising before step a) the following steps: A1) introduction into a culture container a1 comprising a liquid culture medium suitable for the development of said microorganism, i. at least two magnetic or magnetizable particles, ii. at least one microorganism for seeding said medium, and iii. at least one compound and / or physical agent A2) keeping the seeded culture medium and said at least one molecule under conditions permitting a development and / or growth of said microorganism, the medium obtained being the medium introduced into the container of the step at). 3. Method according to any one of the preceding claims, wherein the ligand of said microorganism is selected from the group comprising antibodies, peptides, aptamers. The method of any of the preceding claims, wherein the ligand is attached to the surface of the inner walls of said container at a concentration of 0.1 to 20 μg / ml. The method of any of the preceding claims, wherein the ligand of said microorganism is selected from a monoclonal antibody, a polyclonal antibody or a mixture thereof. The method of claim 5, wherein the concentration of bound antibodies is from 0.5 to 7 μg / ml. 7. Method according to any one of the preceding claims wherein said at least two particles are subjected to an electromagnetic field, optionally applied by pulse. The method of any of the preceding claims, wherein said at least two particles are subject to a gradual increase in the electromagnetic field. The method of any of the preceding claims, wherein said magnetic field is generated by moving field generating means. The method of any one of the preceding claims, wherein said at least two particles are illuminated by means of a light source to sense its motion. 11. A method according to any one of the preceding claims, wherein said particles, at least two, are generating a signal. 12. Method according to the preceding claim, wherein said particles, at least two, are excitable, fluorescent or phosphorescent or radioactive or chemiluminescent. The method of any of the preceding claims, wherein the process is performed with a plurality of particles of 2 to 10,000,000 particles. 14. A method according to any preceding claim wherein the antibiotic is selected from the group consisting of fusidic acid, aminoglycosides, beta-Lactamines, penicillins, beta-lactamase inhibitors, cephalosporins, carbapenems. , monobactams, cycloserine, daptomycin, fosfomycin, lincosamides, macrolides and ketolides, mupirocine, nitroimidazoles, nitrofurans, oligosaccharides, oxazolidinones, polypeptides: bacitracin, glycopeptides, polymyxins and colistin, quinolones, rifamycins, sulfonamides and diaminopyridines, synergistins, tetracyclines, antifungals. 15. The method of claim 1, wherein a plurality of containers comprising on the surface of their inner walls a ligand said microorganism each comprising: said culture medium, said at least two particles, and said microorganism, said compound and or physical agent, said containers comprising a different concentration of said compound and / or physical agent and / or a compound and / or physical agent different from one container to another. 16. The method of claim 15, wherein the plurality of said containers determines the minimum concentration of the antibiotic for said microorganism. 17. Use of the method defined according to any one of claims 1 to 16 for determining the minimum inhibitory concentration of a microorganism of an antibiotic.