METHOD AND SYSTEM FOR DETECTING AND POSSIBLY IDENTIFYING A MICROORGANISM CONTAINED IN A SAMPLE

Abstract

The present invention relates to a method for detecting and possibly identifying a microorganism contained in a sample, comprising the steps of (a) placing the sample in a liquid culture medium, in the presence of a measuring electrode and a reference electrode, under conditions suitable for the growth of microorganisms, (b) measuring, over time, the potential difference between the measuring electrode and the reference electrode, (c) calculating the derivative of the potential difference from the measurements made in step b), thereby obtaining the potential derivative profile, and (d) detecting and possibly identifying a microorganism contained in said sample from the profile obtained in step c). The present invention also relates to a system that can be implemented during this method.

Description

Title of the invention: METHOD AND SYSTEM FOR DETECTING AND POSSIBLY IDENTIFYING A MICROORGANISM CONTAINED IN A SAMPLE Technical field

[0001] The present invention belongs to the technical field of diagnostic microbiology and more particularly to the field of the detection and identification of microorganisms present in a liquid or solid sample.

[0002] The main advance of the present invention lies in the use of electrochemistry not only for the detection of a microorganism such as a pathogen but also and in parallel for its identification. The applications of the present invention relate in particular to sterility tests in the health environment but also in other environments such as the pharmaceutical, biomedical, environmental or agri-food environment. State of the prior art

[0003] In clinical microbiology, and more specifically in the field of blood culture, the operator takes approximately 10 mL of blood from a patient. This test sample is placed in a bottle containing between 30 and 40 mL of a culture medium conducive to bacterial growth. The principle of detection is based on bacterial respiration; by multiplying, the bacteria will produce a detectable quantity of carbon dioxide.

[0004] For example, the bottom of the bottle can be made of a polymer, which will change color or fluorescence depending on the CO2 concentration of the liquid. The blood culture machines then allow the bottles to be shaken at 37°C while using an optical method to check the variation in color or fluorescence of the polymer.

[0005] Once the bottle has been detected as positive, the operator must take a small amount of the culture broth in order to perform a Gram stain. This manipulation is not automated and requires dangerous products such as, for example, crystal violet or gentian violet, Lugol's solution (iodine), alcohol, acetone or fuchsin. After this first orientation, the identification is refined with the use of a MALDLTOF mass spectrophotometer (for "Matrix Assisted Laser Desorption / Ionization-Time Of Flight") and then an antibiogram.

[0006] It is clear from this long multi-step process that the time between the test being taken and the result being given is, at best, several dozen hours. This significant delay cannot be associated with a lack of treatment, broad-spectrum antibiotic therapy is started without waiting for the blood culture results. However, antibiotics that are poorly or not adapted to the pathogen can lead to antibiotic resistance, one of the greatest dangers to global health.

[0007] Automated blood culture systems or CMBCS for "Continuous Monitoring Blood Culture System" are today all systems which are limited to sterility testing only: they detect the presence of a micro-organism, even if it is a single cell, without giving the slightest information as to its nature as eukaryotic or prokaryotic, Gram+ or Gram-, or even species.

[0008] We can cite radiometric respirometry systems (Bactec" 460, BD), infrared CO2 detection (Bactec" 660 / 730 / 860, BD), fluorescence CO2 detection (Bactec" 9240, BD), colorimetric CO2 detection (BacT / ALERT ®, bioMérieux) or manometric detection (VersaTREK ", ThermoScientific and SIGNAL ", ThermoScientific).

[0009] Few blood culture machines have offered both detection and identification, or at least, orientation. We can cite: - in gas phase: the use of a CSA (for “Colorimetry Sensor Array”), an artificial nose with colorimetric transduction, inserted into the bottle cap [1]; - in liquid phase: the use of antibody networks on an SPR prism in contact with the blood culture [2].

[0010] In electrochemistry, it is common to use pH-sensitive electrodes. Several materials are known for their characteristic potential variation in a given pH range, among them, conductive polymers such as polyaniline and metal oxides such as iridium oxide.

[0011] International application WO 2021 / 032654 A1 [3] proposes a device of the autonomous automaton type making it possible to detect the presence of microorganisms in a sample by monitoring, by electrochemistry, the pH variations.

[0012] The majority of research teams working on these technologies use electrochemistry for the a priori detection of a pathogen. Thanks to the use of a probe immobilized on an electrode and by choosing the appropriate measurement method, it is in fact possible to detect the coupling between probe and microorganism.

[0013] The probe depends on the pathogen, the environment and the technique used. It is possible to graft an antibody, a bacteriophage, a thiol probe, an aptamer, a DNA probe, an RNA probe or a protein-specific probe in order to detect a biological mechanism specific to the desired bacterium.

[0014] The main techniques used are amperometry consisting of the detection of a variation in current and impedance spectroscopy consisting of the detection of a modification in the resistance of the electrode.

[0015] The major obstacle to the use of these technologies in the field of diagnostic microbiology for the detection step is their a priori detection, which is unthinkable in clinical terms. In addition, although these types of electrodes make it possible to discriminate between pathogens that are being sought and other "parasitic" microbes, it is not possible to identify a pathogen other than that being sought.

[0016] The inventors set themselves the goal of proposing a method that addresses the technical problems of the methods of the prior art, i.e. a method that is easy to implement, rapid, efficient, making it possible not only to detect but also to identify a microorganism contained in a liquid or solid sample. Presentation of the invention

[0017] The present invention makes it possible to achieve the goal set by the inventors. Indeed, the latter have shown that it is possible to propose a method that is easy to implement, rapid, effective, thanks to which it is possible to detect but also to identify a microorganism contained in a liquid or solid sample, using electrochemistry.

[0018] In particular, the invention is based on a variation of electrochemical signal and in particular on the evolution of the derivative of the electrochemical potential with respect to time of at least one and typically of several electrodes of different materials. With suitable signal processing, it is possible to detect the growth of a microorganism such as bacterial growth and to guide the identification. This therefore allows a reduction in the time before results, a reduction in working time and risks for the operator.

[0019] The method which is the subject of the invention can be implemented on the instrumented bottle as described in international application WO 2021 / 032654 A1 [3] in which electrodes composed of different materials are placed, allowing faster detection for two reasons: 1) the bottle is already instrumented and does not require a bulky automaton, the transport time usually required is no longer necessary and 2) the measurement is carried out directly in the bottle and not via a polymer sensitive to carbon dioxide.

[0020] More particularly, the present invention relates to a method for detecting and possibly identifying a microorganism contained in a sample comprising the steps of a) placing the sample, in a liquid culture medium, in the presence of a measuring electrode and a reference electrode and in conditions suitable for the growth of the microorganisms, b) measure, over time, the potential difference between the measuring electrode and the reference electrode, c) calculating the derivative of the potential difference from the measurement carried out in step b) whereby the profile of the potential derivative is obtained, and d) detecting and possibly identifying a microorganism contained in said sample from the profile obtained in step c).

[0021] The sample used in the context of the method of the invention may be any sample that is usually sterile but may contain microorganisms following a particular event such as contamination, infection, pathology or malfunction of a filtration, decontamination or cleaning system. This sample may be in liquid or solid form.

[0022] The sample used in the method according to the invention is advantageously chosen from the group consisting of a biological fluid; water such as, for example, sterile grade filtered water used in particular in medical installations and production, groundwater, spring water or surface water; a sample from a liquid industrial effluent; a sample from air filtration; a sample from a food, cosmetic or pharmaceutical product and a sample from an object.

[0023] In a particular embodiment, the biological fluid is advantageously chosen from the group consisting of blood such as whole blood or anticoagulated whole blood, blood serum, blood plasma, lymph, tears, sperm, burin, milk, cerebrospinal fluid, interstitial fluid, joint fluid, pericardial fluid, fluid isolated from bone marrow, cell extract, tissue extract, organ extract and one of their mixtures. Thus, the biological fluid can be any fluid naturally secreted or excreted from a human or animal body or any fluid recovered from a human or animal body by any technique known to those skilled in the art such as extraction, sampling, puncture or washing. The steps of recovering and isolating these different fluids from the human or animal body are carried out prior to implementing the method according to the invention.

[0024] In a particular embodiment, the liquid industrial effluent from which the sample is taken may be an effluent from an agri-food, pharmaceutical or cosmetic industry.

[0025] In a particular embodiment, the object from which the sample is taken can be chosen from large installations such as an industrial object such as an electronic device or a machine used in the food, pharmaceutical or cosmetic industries, a tank, a restaurant kitchen, a cold room, a sanitary facility, a container, and small objects such as medical devices or pipes.

[0026] In the context of the present invention, "sampling" means any type of sample collection, for example, by contact, scraping, swabbing, drilling, cutting, punching, grinding, washing, rinsing, suction, puncture or pumping.

[0027] In a more particular embodiment, the sample used in the method according to the invention is blood such as human or animal blood. The latter is normally sterile but may contain microorganisms such as bacteria during non-pathological or pathological bacteremia. Indeed, bacteria may be present in the blood following theoretically harmless acts such as brushing teeth, dental care or medical acts involving probes or catheters. This is then referred to as non-pathological bacteremia. Pathological bacteremia corresponds to the presence of bacteria in the blood following pneumonia, a wound or a urinary infection.

[0028] Among the microorganisms contained or likely to be contained in the sample, there are both prokaryotic microorganisms such as archaea or Gram+ or Gram- bacteria, as well as eukaryotic microorganisms such as yeasts and other microscopic fungi and protists such as algae or protozoa.

[0029] As illustrative and non-limiting examples of microorganisms contained or likely to be contained in the sample used in the context of the invention, mention may be made of bacteria of the family Enterobacteriaceae, bacteria of the family Pseudomonadaceae, bacteria of the genus Staphylococcus, bacteria of the genus Streptococcus, bacteria of the genus Campylobacter, bacteria of the genus Haemophilus, bacteria of the genus Anaerococcus, bacteria of the genus Bacteroides, bacteria of the genus Acinetobacter, bacteria of the genus Stenotrophomonas, bacteria of the genus Achromobacter, bacteria of the genus Fusobacterium, bacteria of the genus Pasteurella, bacteria of the genus Bacillus, bacteria of the genus Listeria, bacteria of the genus Clostridium, bacteria of the genus Mycobacteria, bacteria of the genus Enterococcus and bacteria of the genus Pasteurella, yeasts of the genus Candida, yeasts of the genus Saccharomyces,fungi of the genus Aspergillus, fungi of the genus Penicillium and / or archaea of ​​the genus Methanobrevibacter.

[0030] Furthermore, among the bacteria of the Enterobacteriaceae family contained or likely to be contained in the sample used within the framework of the invention, mention may be made of bacteria of the genera Escherichia, Salmonella, Shigella, Enterobacter, Klebsiella, Serratia, Proteus, Morganella, Yersinia, Citrobacter and Providencia.

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[0035]

[0036] Prior to contact with the liquid culture medium in the presence of the electrodes, i.e. prior to step a) of the method according to the invention, the sample may be subjected to a preparation step by means of any process for preparing a sample likely to contain microorganisms, known to those skilled in the art. This step may in particular consist, in a non-exhaustive manner, of a dilution, a concentration, a division into equivalent or non-equivalent portions or a suspension. Typically, during step a) of the method according to the invention, the sample is diluted in the liquid culture medium, when this sample is liquid. Alternatively, when it is solid, it is suspended in the liquid culture medium. The liquid culture medium used during step a) of the method according to the invention may be any liquid medium suitable for the culture of microorganisms, known to those skilled in the art. By "suitable for the culture of microorganisms" is meant a preparation in which the microorganisms can multiply. This medium comprises a carbon source, such as glucose, acetate or glycerol; a nitrogen source, such as ammonium, nitrates or amino acids; and salts and / or trace elements and vitamins for the growth of microorganisms. In particular, this liquid culture medium may not be selective for a particular type of microorganism. Alternatively, it may be selective for a particular type of microorganism. Examples of liquid culture media that can be used in step a) of the method according to the invention include NB1 culture medium (“Nutrient Broth No. 1”), NB2 culture medium (“Nutrient Broth No. 2”), Mueller Hinton culture medium, LB culture medium (“Lysogen Broth”, also known as “Luria Bertani”), TSB culture medium (“Tryptic Soybean Broth”), BHI culture medium (“Brain Heart Infusion”) and BACT / ALERT® culture medium. These culture media are available in particular from ThermoFisher, Bio-Rad Laboratories, bioMérieux and Sigma Aldrich. In step a) of the method according to the invention, conditions suitable for the growth of the microorganisms are implemented. In other words, during step a) of the method according to the present invention, the microorganisms are maintained in an environment which allows them to increase in number by cell division. This environment includes not only the culture medium in which the sample has been diluted or resuspended, but also other conditions such as a temperature and an atmosphere which allow cell growth. Typically, the temperature used in step a) is between 20°C and 40°C. Particular examples of the temperature used in step a) are 20°C, 25°C, 30°C, 32°C, 37°C and 39°C.

[0037] With regard to atmospheric conditions, step a) can be carried out under an oxygen-free atmosphere, under air, in air + 5% CO2 or under a gas mixture composed of 5% O2, 5% CO2 and 90% N2. An oxygen-free atmosphere, i.e. an anoxic atmosphere, is to be preferred when the microorganism(s) contained or likely to be contained in the sample develop under strict anaerobic conditions. Advantageously, as previously explained, the method according to the invention is carried out in an instrumented bottle as described in international application WO 2021 / 032654 A1 [3], the atmospheric conditions therefore correspond to the air contained in the bottle following its hermetic closure by a stopper.

[0038] During step a) of the method according to the invention, the culture medium comprising the sample likely to contain or containing microorganisms is in contact with at least two electrodes. These electrodes correspond to at least one measuring electrode, also designated by the expression “working electrode” and a reference electrode.

[0039] In a particular embodiment, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten measuring electrodes, identical or different, can be implemented. The experimental part below presents a more particular embodiment with four different pairs of measuring electrodes and a reference electrode.

[0040] By "different measuring electrodes" is meant electrodes whose chemical nature differs. Any measuring electrode known to those skilled in the art can be used in the context of the present invention.

[0041] Thus, the measuring electrode used in the method according to the invention can be a solid metal electrode, a glass electrode, a vitreous carbon electrode, an electrode produced in the form of a deposit of a conductive ink or an electrode produced in the form of an electrodeposition of a conductive element.

[0042] Any conductive ink known to those skilled in the art may be used within the scope of the invention. In a particular embodiment, the conductive ink used is an ink based on metal ions or conductive organic polymers such as polythiophene (PT), polyaniline (PANI) optionally doped with dodecylbenzenesulfonic acid (DBSA), poly(3,4-ethylenedioxythiophene) coupled to sodium poly(styrene sulfonate) (PEDOT:PSS), polypyrrole or polyphthalocyanine. In another particular embodiment, the conductive ink used within the scope of the invention is a carbon ink optionally comprising an additional conductive element.

[0043] By "conductive element" is meant an element chosen from the group consisting of conductive organic polymers such as those listed above, and metal-based compounds such as, for example, a metal oxide such as iridium oxide (IrOx), a metal-based pigment such as Prussian blue (Fe(III) ferrocyanide) or an organic or organo-inorganic catalyst such as cobalt phthalocyanine.

[0044] Any reference electrode conventionally used in electrochemistry can be implemented within the scope of the invention. Advantageously, the reference electrode used within the scope of the invention is produced in the form of a deposit of an Ag / AgCl ink covered with a layer of polymer such as a layer of polyurethane, to prevent the diffusion of silver particles and inhibition of the growth of microorganisms.

[0045] It is also possible to place a counter electrode in contact with the liquid culture medium during the method according to the invention. The presence of a counter electrode is not obligatory.

[0046] Everything described in international application WO 2021 / 032654 A1 [3] regarding the measuring and reference electrodes also applies to the present invention, such as, for example, the two-part organization of the electrodes and the electrical contacts implemented.

[0047] Advantageously, the liquid culture medium and the electrodes are sterile or have been sterilized before any contact with the sample, i.e. prior to step a) of the method. The culture medium may be sterilized using various techniques known in the prior art, such as, but not limited to, the use of aseptic techniques for autoclaving and / or preparing the culture medium. The autoclaving technique may in particular be chosen to sterilize the electrodes, possibly in the presence of the liquid culture medium intended to receive the sample.

[0048] Step b) of the method according to the invention corresponds to monitoring the evolution, in the liquid culture medium, of the potential difference between the measuring electrode and the reference electrode.

[0049] In a particular embodiment, this measurement of the potential difference is done continuously. Indeed, it is possible, using a multi-channel potentiostat, to measure the potential difference between the measuring electrode and the reference electrode continuously (modulo the sampling period of the potentiostat - eg a few ms).

[0050] In another particular embodiment, this measurement of the potential difference is done discontinuously.

[0051] To do this, the potential difference is measured at a plurality of distinct time instants. In other words, step b) consists of measuring the difference of potential between the measuring electrode and the reference electrode at a plurality of time instants whereby potential measurement points are obtained.

[0052] By "plurality of distinct time instants" is meant more than 2, in particular more than 5, in particular, more than 10 and, more particularly, more than 20 distinct time instants. These different time instants can be chosen at regular intervals or at irregular intervals. In a particular embodiment, the measurements are carried out at regular intervals. A person skilled in the art will be able to determine, without inventive effort, the most suitable time interval depending on the sample to be studied and the microorganisms likely to be present in the latter. As a particular example, this interval can be between 1 min and 30 min, in particular between 2 min and 15 min and, in particular, of the order of 5 min (5 min ± 1 min).In another form of particular implementation, the measurements are carried out at irregular intervals. Indeed, one can consider taking a measurement controlled by the temporal drift of the signal, thus making it possible to measure more points in the areas of strong variation versus the periods of stagnation of the potential.

[0053] In a particular embodiment and whether the measurement is continuous or discontinuous, it is possible to carry out the first measurement of the potential difference at the time instant ti just after step a) of the method of the invention, i.e. 1 min, 2 min, 3 min, 4 min or even 5 min after the sample has been brought into contact with the liquid culture medium in the presence of the electrodes.

[0054] In another particular embodiment and whether the measurement is continuous or discontinuous, it is possible to carry out the first potential measurement at the time instant ti at least 1 h after the sample has been brought into contact with the liquid culture medium in the presence of the electrodes.

[0055] The interval between the time instant L of the first measurement and the time instant td of the last potential measurement during step b) is variable and depends in particular on the sample to be studied, the microorganisms likely to be present in the latter and the time instant tb. Advantageously, the time instant ta of the last potential measurement is carried out at least 14 h, at least 16 h, at least 18 h, at least 20 h or at least 22 h after step a).

[0056] Advantageously, the operating conditions during step b) in terms of temperature and atmospheric conditions are identical to those implemented during step a) of the process according to the invention as previously described.

[0057] Furthermore, the culture medium may be subjected to agitation during steps a) and / or b) of the method according to the invention. This agitation during step a) allows homogeneous mixing of the sample with the culture medium. During step b) and in particular in the presence of microorganisms, this agitation promotes a composition uniformity of the culture medium and prevents possible deposition of microorganisms on the electrodes. Any known technique for stirring a medium and not interfering with the measurement of the potential can be used within the framework of the present invention. This stirring is in particular as envisaged in international application WO 2021 / 032654 A1 [3],

[0058] Step c) consists of calculating, continuously (case of a measurement of the continuous potential difference during step b)) or from the different potential measurements obtained in step b) (case of a measurement of the discontinuous potential difference during step b)), the derivative of the potential.

[0059] Indeed, in order to observe more clearly the start of the potential variations when microorganisms from the sample develop in the culture medium, the derivative of the potential difference for each electrode is used. Thus, the speed of modification of the potential is observed, much more meaningful and readable than the raw potentiometric curves.

[0060] In a particular embodiment and when the measurement of the potential difference during step b) is discontinuous, the calculation of the derivative of the potential difference can be done in a sliding manner over a predetermined number of measurement points obtained in step b). In other words, the derivative of the smoothed potential difference is calculated over a predetermined number of potential difference measurement points (sliding average over a predetermined number of potential difference measurement points).

[0061] Typically, the pre-determined number of measurement points is equal to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. Advantageously, the pre-determined number of measurement points is equal to 5, in particular when the interval between two consecutive potential measurement points is of the order of 5 min.

[0062] By "sliding manner" is meant that the calculation of the derivative is carried out for a pre-determined number n of potential measurement points by calculating the first value of the derivative corresponding to the average of the derivatives of the potential measurement points from pi to pn, the second value of the derivative corresponding to the average of the derivatives of the potential measurement points from p2 to pn+[, the third value of the derivative corresponding to the average of the derivatives of the potential measurement points from p3 to pn+ 2, and so on...

[0063] Step c) of the method according to the invention can be carried out once step b) has been carried out, i.e. for a discontinuous measurement, once all the potential measurement points have been obtained. Alternatively, step c) of the method according to the invention can be carried out at the same time as step b) of the method and, for a discontinuous measurement, once the first predetermined number of potential measurement points have been obtained. The calculation carried out in step c) generates an n-dimensional response corresponding to the profile of the derivative of the potential difference. From this n-dimensional response, it is possible to extract a normalized profile using methods such as PCA (for "PluriComponent Analysis" or principal component analysis), MDS (for "MultiDimensional Scaling" i.e. multidimensional positioning) and neural networks or Fourier transform to obtain a reduced dimensionality. In other words, the method according to the invention comprises an additional step consisting of obtaining a normalized profile of the derivative of the potential from the profile of the derivative obtained in step c).

[0064] The detection of the presence or absence of microorganisms in the sample can be done by comparing the profile of the derivative of the potential or possibly the normalized profile of the derivative of the potential difference with the profile of the derivative of the potential or possibly the normalized profile of the derivative of the potential difference obtained with a negative control, i.e. devoid of microorganisms and under the same operating conditions and in particular the same working and reference electrode(s) and the same culture medium.

[0065] The identification of a particular microorganism can be done by comparing the profile of the derivative of the potential difference or possibly the normalized profile of the derivative of the potential with different profiles of the derivative of the potential or possibly different normalized profiles of the derivative of the potential difference obtained, each, with a known microorganism and under the same operating conditions and in particular the same working and reference electrode(s) and the same culture medium.

[0066] The present invention also relates to a system for detecting and possibly identifying a microorganism contained in a sample capable of being implemented during the method as previously defined comprising i) a device configured for cell culture in a liquid medium comprising a measuring electrode and a reference electrode, ii) a unit for measuring the potential difference between the measuring electrode and the reference electrode and configured to measure the potential difference, continuously and / or at a plurality of time instants, iii) an electronic unit for acquiring the measurement of the potential difference, connected to the potential measurement unit, iv) a power supply unit for powering the electronic acquisition unit, and v) a processing unit connected to the electronic acquisition unit and configured to process the measurement of the potential difference and calculate, from the latter,the derivative of the potential difference and comprising a module for detecting and possibly identifying a microorganism by analyzing the derivative of the potential difference.

[0067] In a particular embodiment, the processing unit is configured to process the measurement points of the potential difference and calculate, from the latter, the derivative of the potential difference and / or configured to process the continuous measurement of the potential difference and calculate, from the latter, the derivative of the potential difference.

[0068] Other characteristics and advantages of the present invention will become apparent to those skilled in the art upon reading the examples below given for illustrative and non-limiting purposes, with reference to the appended figures. Brief description of the figures

[0069] [Fig-1] presents the potentiometric curves obtained in human blood after inoculation, at t=0, of 38 cfu (for “colony-forming units”) of Escherichia coli - ATCC 8739 (PANI = polyaniline / IrOx = iridium oxide / P2 = SunChemical Gwent C2070424P2 ink / BQ = Dupont BQ242 ink).

[0070] [Fig.2] shows the potentiometric curves obtained in human blood in the absence of any microorganism (PANI = polyaniline / IrOx = iridium oxide / P2 = SunChemical Gwent C2070424P2 ink / BQ = Dupont BQ242 ink).

[0071] [Fig.3] shows the smoothed derivatives of the potential of the polyaniline-based electrode, obtained in human blood after growth of different bacteria and for a negative control.

[0072] [Fig.4] presents the smoothed derivatives of the potential of the polyaniline-based electrode, obtained in human blood after growth of all the bacteria tested, time normalized to center the maxima.

[0073] [Fig.5] presents the 3D space allowing the orientation of the identification of bacteremia.

[0074] [Fig.6] presents the 2D space allowing the orientation of the identification of bacteremia.

[0075] [Fig.7] presents the modulus as a function of frequency, calculated from the Fourier transform of the smoothed derivative of the potential of the polyaniline-based electrode, obtained in human blood after growth of all the bacteria tested.

[0076] [Fig.8] shows the correct Gram typing rate (%) versus additional incubation time after culture positivity. Description of the embodiments I. Manufacture of the device used.

[0077] In order to obtain several electrochemical signals and therefore several electrochemical signatures for each pathogen, several electrode materials were used.

[0078] The measuring device is a PCB (for "Printing Circuit Board") platform, with 10 gold electrodes: 8 working electrodes and a reference electrode (. The materials, in the form of ink, are deposited on the gold then dried in an oven, at 80°C, for 2 h. The measuring device can optionally include a counter-electrode such as a counter-electrode made of a commercial carbon ink (Dupont BQ242).

[0079] 1.1. Working electrodes.

[0080] Each electrode is made in duplicate on the platform in order to avoid possible problems with a deposit. There are therefore 4 different inks: i) polyaniline (PANI), doped with dodecylbenzenesulfonic acid (DBSA) (Pani-DBSA ink from Rescoll); ii) iridium oxide (IrOx), a metal oxide, incorporated into a carbon ink (Dupont BQ242); (iii) SunChemical Gwent C2070424P2, commercial carbon ink with Prussian blue (Fe(III) ferrocyanide); and iv) commercial carbon ink (Dupont BQ242).

[0081] PANI and IrOx inks have a known and stable sensitivity to pH variations while SunChemical Gwent C2070424P2 and Dupont BQ242 are chosen for the detection of other chemical reactions within the medium.

[0082] Each working electrode has an undercoat of Dupont BQ242 carbon ink to improve the mechanical strength of the deposit.

[0083] 1.2. Reference electrode.

[0084] On the gold electrode provided for this purpose, an underlayer of Dupont BQ242 carbon ink and then a layer of Ag / AgCl ink are deposited. Ag / AgCl ink is commonly used as a material for pseudo-reference electrodes.

[0085] This electrode being particularly sensitive to oxidative stress induced by the sterilization method used (autoclave), it is also protected by a polyurethane membrane which is left to dry for 24 hours, at room temperature, after deposition.

[0086] 1.3. Manufacture of the bottle.

[0087] Once the platform is prepared as explained above, it is integrated into an Azlon® polypropylene bottle. The slot used to pass the connectors outside the bottle is sealed by three successive methods: - addition of LOCTITE® 406™ cyanoacrylate glue, after application of an activator,

[0088] - addition of RS PRO fast-setting epoxy glue, and - masking of glue joints with Kapton® adhesive tape.

[0089] Thanks to these precautions, the bottle is completely watertight and experiments without risk of contamination are possible. II. Preparation of the experiment.

[0090] II. 1. Prior sterilization.

[0091] Once the bottle is filled with approximately 30 mL of BACT / ALERT® culture medium, it is autoclaved in a Tuttnauer 2540 EL autoclave using a “Liquid” cycle. It is then subjected to a temperature increase to a plateau at 121°C for 15 min. This method ensures the sterility of the electrodes and the culture medium.

[0092] II.2. Seeding protocol.

[0093] Under Microbiological Safety Cabinet (MSC), 10 mL of blood is added to the bottle. If the chosen experiment is negative (without bacteria) then the measurement is started.

[0094] Otherwise, the bottle is inoculated with a certain quantity of bacteria following the protocol explained below, all manipulations taking place under PSM with the safety measures in force.

[0095] Bacterial colonies are added to a bottle filled with 3 mL of pharmaceutical diluent (European Pharmacopoeia Diluent - 9 mL tube, VWR AX011144). The turbidity of this mixture is regularly measured by a turbidimeter to reach a value of 0.5 McFarland.

[0096] The addition of colonies is carried out using a loop (or inoculation loop) from a culture on a Petri dish which has been prepared and then placed at 37°C for 24 hours.

[0097] Once the value of 0.5 McFarland is reached, a cascade dilution is carried out in pharmaceutical diluent until the desired theoretical bacterial concentration is reached.

[0098] A volume of 0.4 mL of this last dilution is taken and then added to the measuring bottle previously filled with 30 mL of BACT / ALERT® culture medium and 10 mL of blood. This is therefore a 1 / 100th inoculation.

[0099] IL 3. Counting protocol.

[0100] In order to ensure the actual quantity inoculated at t = 0s in the device, counts on dishes are carried out. For this, a volume of 0.1 mL of a dilution is taken and then spread on a Petri dish with a rake.

[0101] This counting is carried out on the last two dilutions, in triplicate in order to be able to take an average. III. The measurement method.

[0102] The electrochemical measurement method used is potentiometry, i.e. a potential difference between a working electrode and a reference electrode whose potential does not vary over time.

[0103] The device being composed of 8 working electrodes, the measurement is multiplexed: every 5 minutes, the potential of each electrode is recorded for 0.1s.

[0104] This method allows to obtain 8 potential curves as a function of time. The experiment was repeated with several different bacteria, several initial quantities and even without any bacteria in order to obtain negatives providing information on the stability of the measurement.

[0105] [Fig.l] shows the potentiometric curves obtained after inoculating, at t = 0, human blood with 38 cfu of Escherichia coli - ATCC 8739. Two characteristic potential profiles are observed, a “polyaniline” profile where the potential increases then slowly decreases, and a “carbon ink” profile where the profile drops more or less abruptly.

[0106] It is clear, in view of the measured values, that it is not only the variation in the pH of the measuring medium which is detected but other electrochemical reactions, probably due to the secretion of redox metabolites. IV. Exploitation of signals.

[0107] Since the biological media used, such as blood, are not inert, the electrodes record potential variations even without the presence of bacteria.

[0108] For a 5-day negative control, which corresponds to the standard time in a hospital environment to determine the absence of bacteria, a slow drift in the potential is observed for all the electrodes (see [Fig.2]). These variations are very small compared to experiments with bacteria.

[0109] In order to observe more clearly the beginning of the potential variations in cases of bacteremia, the derivative of the potential smoothed over 5 points (sliding average over 5 points) for each electrode is used. Thus, the speed of modification of the potential is observed, much more meaningful and readable than the raw potentiometric curves.

[0110] Advantageously, the smoothed derivative of the potential is only looked at from 4 hours of recording. Indeed, bacteria have an incompressible latency time during which any detection is unthinkable because bacterial growth has not yet begun.

[0111] The curves presented in [Fig.3] are therefore derivatives (smoothed over 5 points) of the potential, from t = 4h. As illustrated in this figure, the detection is clearly visible and it is possible to calculate a detection time.

[0112] In addition, here with the polyaniline electrode, we notice a characteristic profile of the curve as a function of the bacteria detected. This difference in profile being repeatable, it is possible to use it to guide the identification of bacteremia in parallel with its detection.

[0113] The curve profile differences are visible on all electrodes but polyaniline seems the most suitable for identification, the differences being more

[0114]

[0115]

[0116]

[0117] obvious. On the other hand, detection is clear for all electrodes, even faster for carbon ink-based electrodes: iridium oxide, SunChemical Gwent C2070424P2 and Dupont BQ242. V. Creating a descriptor. A descriptor was created from the different curves measured on polyaniline electrodes. However, it is quite possible to create a descriptor for each electrode. This descriptor makes it possible to determine the bacterial species based on the profile of the curve of the smoothed derivative of the potential. To do this, we use a database obtained from experiments carried out on human blood with different bacteria. The details of these experiments are provided in Table 1 below. [Tables 1] Bacteria Strain Inoculum at t = 0 in cfu Escherichia coli ECU-ATCC 2739 10 10 100 100 EC21 - ATCC 35421 10 10 100 100 EC10-ATCC 25922 10 10 100 100 Staphylococcus epidermidis SE26 - ATCC 12228 10 10 100 100 SE97 - ATCC 35984 10 10 100 100 SE101 -ATCC NCIMB 8853 10 10 100 100 Klebsiella aerogenes KA31 - ATCC 13048 10 10 100 100 Enterobacter cloacae EC8 - ATCC 13047 10 10 100 100 Citrobacter freundii CF7 - ATCC 8090 10 10 100 100

[0118] For each experiment, we have two polyaniline signals because there are two polyaniline electrodes per device. This therefore represents a total of 36 smoothed derivatives of the potential as a function of time.

[0119] We start by removing the time dependence of the curves. By translation, the detection times are placed at the same time (see [Fig.4]). We therefore obtain, for each curve, a certain number of points (1 point every 5 minutes from 8 p.m. to 12 a.m. in [Fig.4]).

[0120] From this data, the Tanagra software can be used to perform an unsupervised principal component analysis (PCA). This analysis makes it possible to represent the different points in a 3D ([Fig.5]) or 2D ([Fig.6]) space.

[0121] In [Fig.5], we observe very distinct zones for each bacterial group, confirming the relevance of this descriptor as to its use to guide the identification of the detected pathogen.

[0122] With such a database to be completed with other bacteria, it is possible to discriminate Gram+ bacteria from Gram- bacteria, and it is possible to identify the bacterial species.

[0123] In order to reduce the number of dimensions of the vector, it is also possible to apply a Fourier transform to each curve presented [Fig.4]. Thus, we obtain, for each experiment, 17 data corresponding to the 17 frequencies of interest between 0.1 and 2 Hertz as shown [Fig.7].

[0124] A fairly clear difference is observed between the different groups of bacteria: E.coli / Other enterobacteria (KA31, EC8 and CF7) / S.epidermidis.

[0125] Thanks to this data, it is possible to do supervised learning (or "support vector machine") in order to obtain a confidence index regarding the determination of the Gram of the bacteria as a function of time after detection ([Fig.8]).

[0126] The method according to the invention therefore makes it possible to determine the Gram of the bacteria without any other manipulation or any product, unlike the hospital reality where Gram staining is done under a microscope after adding chemical products. Thirty minutes after detection, there is an accuracy of 82%, 2 hours after detection, 90% accuracy and, 3 hours after detection, 99% accuracy. The more information obtained, the more it is possible to specify the nature of the pathogen detected. Bibliographic references

[0127] [1] Shrestha et al, 2017, “The combined rapid detection and species-level identification of yeasts in simulated blood culture using a colorimetry sensor array”, PLoSONE 12(3): e0173130. doi:10.1371 / joumal.pone.0173130

[0128] [2] Templier et al, 2017, “Biochips for Direct Detection and Identification of Bacteria in Blood Culture-Like Conditions”, 7(1):9457. doi: 10.1038 / s41598-017-10072-z.

[0129] [3] International application WO 2021 / 032654 Al in the name of the Commission à Atomic Energy and Alternative Energies, published on February 25, 2021.

Claims

Claims

1. A method for detecting and identifying a microorganism contained in a sample comprising the steps of a) placing the sample, in a liquid culture medium, in the presence of a measuring electrode and a reference electrode and under conditions suitable for the growth of the microorganisms, b) measuring, over time, the potential difference between the measuring electrode and the reference electrode, c) calculating the derivative of the potential difference from the measurement carried out in step b) whereby the profile of the potential derivative is obtained, and d) detecting and identifying a microorganism contained in said sample from the profile obtained in step c).

2. Method according to claim 1, characterized in that said sample is chosen from the group consisting of a biological fluid; water such as, for example, sterile grade filtered water used in particular in medical installations and production, groundwater, spring water or surface water; a sample from a liquid industrial effluent; a sample from air filtration; and a sample from an object.

3. Method according to claim 1 or 2, characterized in that said sample is blood.

4. Method according to any one of claims 1 to 3, characterized in that said microorganism is chosen from the group consisting of prokaryotic microorganisms such as archaea or Gram+ or Gram- bacteria, eukaryotic microorganisms such as yeasts and other microscopic fungi and protists such as algae or protozoa.

5. Method according to claim 4, characterized in that said measuring electrode is a solid metal electrode, a glass electrode, a glassy carbon electrode, an electrode produced in the form of a deposit of a conductive ink or an electrode produced in the form of an electrodeposition of a conductive element.

6. Method according to claim 4 or 5, characterized in that said reference electrode is produced in the form of a deposit of an Ag / AgCl ink covered with a layer of polymer.

7. Method according to any one of claims 1 to 6, characterized in that said measurement of the potential difference during step b) is carried out continuously.

8. Method according to any one of claims 1 to 6, characterized in that said measurement of the potential difference during step b) is carried out discontinuously, said step b) consisting of measuring the potential difference between the measuring electrode and the reference electrode at a plurality of time instants whereby potential measurement points are obtained.

9. Method according to any one of claims 1 to 8, characterized in that said culture medium is subjected to agitation during said steps a) and / or b).

10. Method according to any one of claims 1 to 9, characterized in that said method comprises an additional step of obtaining a normalized profile of the derivative of the potential from the profile of the derivative obtained in step c).

11. System for detecting and identifying a microorganism contained in a sample capable of being implemented during the method as defined in any one of claims 1 to 9 comprising i) a device configured for cell culture in a liquid medium comprising a measuring electrode and a reference electrode, ii) a unit for measuring the potential difference between the measuring electrode and the reference electrode and configured to measure the potential difference, continuously and / or at a plurality of time instants, iii) an electronic unit for acquiring the measurement of the potential difference, connected to the potential measurement unit, iv) a power supply unit for powering the electronic acquisition unit, and v) a processing unit connected to the electronic acquisition unit and configured to process the measurement of the potential difference and calculate, from the latter,the derivative of the potential difference and comprising a module for detecting and identifying a microorganism by analyzing the derivative of the potential difference.,

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