Method for detecting and identifying microorganisms in a sample by fragmentation of the sample

Abstract

The invention relates to a method for detecting the presence of several microorganisms and identifying said microorganisms in the same sample, the method comprising the steps of: Fragmentation (E2) of the sample (ECH) into several subsamples; Placement (E3) of each subsample (S1-S10) in a separate compartment (C1-C10), said compartments containing the same culture medium and each being instrumented with a measuring electrode (E10) and at least one reference electrode (E20); Measurement (E4) over time in each compartment of the potential difference between the measuring electrode (E10) and the reference electrode (E20); Determination of the derivative profile of the potential difference for each subsample (S1-S10); Detection (E5) of the presence or absence of each microorganism present in each subsample (S1-S10) and identification of the nature of each microorganism present,(from the profile obtained in step d). Figure to be published with the abbreviation: Figure 1,

Description

Title of the invention: Method for detecting and identifying microorganisms in a sample by fragmentation of the sample Technical field of the invention

[0001] The present invention relates to a method for detecting the presence of at least one microorganism within a sample, by fragmenting the sample into several sub-samples. The invention also relates to a detection system capable of implementing the method. State of the art

[0002] Blood is normally completely sterile. Any microorganism present in the blood system therefore represents a vital threat to the human body. Blood culture is currently a known method for diagnosing blood infections. It is a common test in clinical microbiology. Blood culture consists first of all in culturing blood in a nutrient broth under aerobic and anaerobic conditions, under incubation at 36-37°C in automated systems adapted to this type of samples, in order to obtain bacterial growth. The objective is to amplify the quantity of bacteria present in the sample, by providing it with favorable growth conditions, and thus to detect the presence of bacteria in the blood of a patient with a septic condition.This amplification is all the more necessary since the bacterial concentration in the blood during bacteremia is always low, of the order of one or a few bacteria per milliliter of blood taken. This first stage of blood culture therefore consists of a simple detection of presence, without identification, from a sample without normal flora: any presence of bacteria or fungus therefore leads to a positive test.

[0003] This detection will then be followed by a culture on agar medium to isolate the pathogen in the form of colonies, then allowing the identification of the pathogen and its antibiogram to be carried out. In the case of blood infections, the tests are urgent and the time taken to return the results (positive blood culture, identity of the pathogen, antibiogram) has an undeniable impact on the outcome of patients (mortality, length of hospitalization, complications, etc.). The blood culture results allow antibiotic therapy to be adapted to the specific case of the patient: the earlier appropriate and effective antibiotic therapy is initiated, the greater the patient's chances of survival. Each hour of delay in initiating appropriate antibiotic therapy is associated with an increase in mortality.

[0004] Diagnostic manufacturers have worked for many years to reduce the positivity time of blood cultures, as well as to reduce analysis times on identification and antibiogram tests. Currently, two main types of machines coexist: - Devices that monitor the amount of carbonic acid generated in the liquid phase, using a polymer matrix (silicone) loaded with a pH-sensitive chromophore or fluorophore. - Devices used to detect an increase in total pressure in the gas phase.

[0005] These two technologies have in common that they are implemented in automated machines, often large, with the impossibility of starting the test until the bottle is in the machine (pre-incubation can generate false negatives), hence the loss of precious hours before the implementation of optimized antibiotic therapy. A study in 2013 showed that the average transport time was 9 h (interquartile range: 3-15 h), with 6% of bottles having a transport time greater than 20 h.

[0006] Patent application EP4018191A1 describes the use of an instrumented container equipped with measuring electrodes, capable of receiving a liquid sample to be analyzed. The detection of the presence of microorganisms can be carried out in particular by electrochemistry, by placing the container in a suitable box. This patent application proposes a simple solution for reducing the time between taking the sample and starting the analysis of the sample taken. This solution is also easily transportable, making it available in the field, and easy to implement, even by unskilled personnel.

[0007] A recent method (patent application FR2114688) makes it possible to detect and identify the growth of a microorganism in a liquid sample using potentiometric signatures recorded over time. This method is particularly suitable when a single bacterial species is present in the sample and allows for a shorter detection time when the bacterial concentration is higher.

[0008] On the other hand, in the case of polymicrobial contamination (at least two different species of microorganisms in the same sample), the electrochemical signatures generated only make it possible to identify the species which multiplies most rapidly, the colonization of the electrodes then preventing the appearance of a new potentiometric signature indicating the presence of a second species.

[0009] The aim of the invention is to propose a method for making a sample taken more readable, in particular when the contamination is polymicrobial. Presentation of the invention

[0010] This goal is achieved by a method of detecting the presence of several micro organisms and identification of said microorganisms in the same sample, the method comprising steps of: a. Fragmentation of the sample into several sub-samples, b. Placement of each sub-sample in a separate compartment, said compartments containing the same culture medium and each being instrumented using a measuring electrode and at least one reference electrode, c. Measurement over time in each compartment of the potential difference between the measuring electrode and the reference electrode, d. Determination of the profile of the derivative of the potential difference for each sub-sample, e. Detection of the presence or absence of each microorganism present in each sub-sample and identification of the nature of each microorganism present, from the profile obtained in step d).

[0011] According to a particular feature, for each sub-sample, the method 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 d).

[0012] According to another feature, said sample is blood.

[0013] The invention also relates to a detection system, suitable for implementing the method as defined above, comprising a measuring device with several compartments, each compartment being intended to receive a distinct sub-sample, each compartment being instrumented using at least one measuring electrode and one reference electrode.

[0014] According to a particular embodiment, the measuring device is in the form of a single bottle whose internal volume comprises partitions arranged to form said compartments.

[0015] According to one feature, the bottle has a common inlet and a distributor having several flow channels each opening into a separate compartment of the bottle.

[0016] According to another particular embodiment, the measuring device comprises a well plate, each well forming a separate instrumented compartment.

[0017] According to a particular feature, the well plate comprises a first plate provided with through holes against which is applied a second plate carrying, for each through hole, a measuring electrode and a reference electrode.

[0018] According to another feature, each measuring electrode is produced in the form of a deposit of a conductive ink or an electro-deposition of a conductive measuring element.

[0019] According to another feature, said reference electrode is produced in the form of a deposit of an Ag / AgCl ink covered with a polymer layer.

[0020] The microorganism is for example 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. Brief description of the figures

[0021] Other characteristics and advantages will appear in the detailed description which follows, given with reference to the appended drawings in which: - [Fig.l] illustrates the principle of carrying out the method of the invention; - [Fig.2] illustrates the principle of fragmentation of a sample; - [Fig.3] illustrates the principle of fragmentation of a sample and allows highlight the detection of two bacteria A and B within the fragments of this sample; - Figures 4A and 4B show a first example of embodiment of the measuring device of the invention, respectively seen from the side and seen from above in a cross-section; - Figures 5A and 5B show a second example of embodiment of the measuring device of the invention, respectively seen in perspective and seen from above;

[0022] Detailed description of at least one embodiment

[0023] The invention aims in particular at a method which is easy to implement, rapid and effective, thanks to which it is possible to detect but also to identify the micro-organisms contained in a liquid or solid sample, using electrochemistry.

[0024] This method is based in particular on the principle of fragmenting the sample into several sub-samples.

[0025] For detection, the invention relies on a variation of electrochemical signal and in particular on the evolution of the derivative of the electrochemical potential with respect to time. With suitable signal processing, it is possible to detect the growth of a microorganism such as bacterial growth and to guide the identification.

[0026] The ECH sample may be a biological fluid, advantageously selected from the group consisting of blood such as whole blood or anticoagulated whole blood, blood serum, blood plasma, lymph, tears, semen, cerebrospinal fluid, interstitial fluid, joint fluid, pericardial fluid, fluid isolated from bone marrow, cell extract, tissue extract, organ extract and a mixture thereof. Thus, the biological fluid may 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 than an extraction, a sample, a puncture or a washing. The steps of recovery and isolation of these different fluids from the human or animal body are carried out beforehand and are not part of the invention.

[0027] The ECH sample may also be a liquid product from a food, pharmaceutical or cosmetic industry. In a particular embodiment, the object from which the sample is taken may 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.

[0028] The ECH sample can also be a sterile culture medium, surrounding a solid or powdered sample whose sterility is to be tested, for example a biopsy, tissues for grafts, a medical device (heart valve, prosthesis, contact lens, syringe, needle, etc.), a powdered active ingredient, single-use laboratory material, surgical equipment, etc.

[0029] In a more particular embodiment, the ECH sample is liquid and consists of blood, such as human or animal blood. The latter is normally sterile but may contain microorganisms such as bacteria.

[0030] Indeed, bacteria can be present in the blood following theoretically harmless acts such as brushing teeth, dental care or medical procedures involving probes or catheters. This is called non-pathological bacteremia. Pathological bacteremia corresponds to the presence of bacteria in the blood following pneumonia, a wound or a urinary infection.

[0031] Among the microorganisms contained or likely to be contained in the sample, there are 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.

[0032] 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 Pseudomonadaceae family, 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 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 Sac- charomyces, fungi of the genus Aspergillus, fungi of the genus Penicillium

[0033] Furthermore, among the bacteria of the order Enterobacterales 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.

[0034] With reference to [Fig.l], the method of the invention comprises the steps described below. First step El

[0035] The first step E1 consists of taking an ECH sample to be analyzed. For example, 10 mL of blood is taken from a patient. Second stage E2

[0036] The second step E2 consists of fragmenting the ECH sample taken into several sub-samples S1, S2, S3, S4, S5, S6, S7, S8, S9, S10.

[0037] Fragmentation of the ECH sample into several sub-samples S1-S10 makes it possible to reduce the detection time, by increasing the microbial concentration in a fragment (by reducing the volume). It also has the advantage of making the ECH sample more readable in the case of a polymicrobial infection.

[0038] Sample fragmentation also allows for a reduction in detection time by recording negatives (sub-samples without bacteria) relating to the patient. This provides a personalized signal baseline, allowing for finer / faster detection.

[0039] [Fig.2] illustrates the classic case of a blood culture. In this case, the bac load The average blood culture volume of a patient with bacteremia is 1 cfu / mL (cfu = "colony forming unit"). For a blood culture, the sample is 10mL. It therefore potentially contains 10 cfu.

[0040] By fragmenting the 10 mL of blood here into 10 volumes of 1 mL, we obtain a disparity of concentrations with two extreme situations: - The bacteria are distributed perfectly equally, - The bacteria are all in the same compartment.

[0041] In both cases, the reduction in volume will allow a reduction in detection time (microbial growth following an exponential law at the time of detection).

[0042] In the majority of cases (therefore excluding extreme cases), we find more concentrated fragments and others without bacteria at all (as can be seen in [Fig.2]).

[0043] If the patient has a polymicrobial infection (at least two bacterial species), fragmentation of the ECH sample is also an advantage: with suf By means of divisions, it is possible to separate the two species and thus reduce the detection to the classic case of a mono-microbial infection.

[0044] The number of fragments in the ECH sample is a piece of data to be studied statistically, to maximize the chances of separating two species during a polymicrobial infection. From a statistical point of view, the number of fragments / divisions must be chosen so that the probability of having 2 cfu in the same sub-sample is very low (of the order of 1%). Thanks to the Poisson distribution, we know that ten divisions are necessary (at least) for a bacterial concentration of the test sample of 1 cfu / mL.

[0045] [Fig.3] illustrates this principle in the case of a sample taken containing a first bacterium A (5 cfu) and a second bacterium B (5 cfu). This ECH sample is fragmented into ten sub-samples S1-S10. [Fig.3] shows an example of the possible distribution of the two bacteria in the ten sub-samples created. We note that: - A subsample S6 contains only bacteria A; - A subsample S1 contains only bacteria B; - Some subsamples S2, S3, S5, S7, S9, S10 do not contain any bacteria (negative); - Some subsamples S4, S8 contain both bacteria A and B (no identification possible); Third stage E3

[0046] Each sub-sample S1-S10 is placed in a separate compartment C1-C10 of a measuring device, each compartment containing a liquid culture medium. Examples of embodiments of a compartmentalized measuring device are given below, in connection with [Fig.4A], [Fig.4B], [Fig.5A] and [Fig.5B].

[0047] The liquid culture medium is identical for all compartments C1-C10 and therefore for all sub-samples S1-S10. The compartments are sealed from each other.

[0048] Each compartment C1-C10 is instrumented using electrochemical measuring means.

[0049] The measuring means comprise at least two different electrodes E10, E20, used to analyze the sub-sample placed in the compartment, a measuring electrode E10 and a reference electrode E20.

[0050] The electrodes E10, E20 are for example integrated into the compartment so that the liquid contained therein comes into contact with the electrodes during the analysis.

[0051] The measuring electrode E10 used is advantageously an electrode produced in the form of a deposit of a conductive ink or an electrode produced in the form of an electro-deposition of a conductive measuring element. Any conductive ink known to a person skilled in the art can be implemented. 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 in the context of the invention is a carbon ink optionally comprising an additional conductive measuring element.By "conductive measuring element" is meant a measuring element selected 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) 5 ferrocyanide) or an organic or organo-inorganic catalyst such as cobalt phthalocyanine.

[0052] Advantageously, the reference electrode E20 used in the context of the invention is produced in the form of a deposit of an Ag / AgCl ink.

[0053] The compartmentalized measuring device is integrated into a detection system configured to detect and optionally identify the microorganisms contained in each sub-sample contained in each compartment.

[0054] The system also includes: - A unit U1 for measuring the potential difference between the measuring electrode E10 and the reference electrode E20 and configured to measure the potential difference in each compartment, continuously and / or at a plurality of time instants, - An electronic acquisition unit U2 for measuring the potential difference, connected to the measuring unit, - A processing unit U3 connected to the electronic acquisition unit U2 and configured to process the measurement of the potential difference, - A U4 power supply unit to power the U2 electronic acquisition unit and possibly the U3 processing unit.

[0055] A heating unit may also be added, as well as a temperature measurement and management unit. Heating must be carried out uniformly, all around the container. The temperature measurement and management unit may include a temperature probe and means for regulating said temperature to a determined fixed value. The temperature value is chosen according to the type of culture implemented. In the case of a blood culture, the incubation temperature is chosen to be equal to 35°C + / - 2°C.

[0056] The power supply unit U4 may consist of a rechargeable battery intended to power the different units of the device.

[0057] The processing unit U3 may comprise a microprocessor and storage means. It may also comprise a communication module. This module may be wireless to communicate via a wireless link, for example according to the Bluetooth protocol, with the corresponding module of the acquisition unit.

[0058] For example, the processing unit U3 can be configured to: - Receive the potential difference measurement data from the acquisition unit, for example via the wireless link, - Establish electrochemical monitoring of the mixture as a function of time, - Calculate the derivative of the acquired potential difference, - Process the curve obtained to determine the presence of microorganisms, for example in. - Control a human-machine interface designed to indicate the presence of bacteria, - Read an RFID tag present on container 1 in order to collect and store data linked to the sample, - Ensure regulation of the heating unit to ensure the most constant incubation temperature possible.

[0059] The electrochemical signature obtained for each compartment will make it possible to identify whether it is a mono-microbial division (classic signature), a negative division (no detection) or a polymicrobial division (noisy signature). Fourth stage E4

[0060] This step E4 of the method corresponds, for each Cl-CIO compartment, to monitoring the evolution, in the liquid culture medium, of the potential difference between the measuring electrode E10 and the reference electrode E20.

[0061] The measurement of the potential difference is done continuously or discontinuously. In the discontinuous mode, the potential difference is measured at a plurality of distinct time instants.

[0062] The interval between the time instant Tl of the first measurement and the time instant Td of the last potential measurement 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 tl. Advantageously, the time instant td 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 the start of the third step E3. Fifth step E5

[0063] This step is implemented by the processing unit U3 and consists of calculating the derivative of the measured electrochemical potential. The U3 processing unit performs this calculation for each compartment and therefore for each subsample.

[0064] Indeed, in order to observe more clearly the start of the potential variations when microorganisms from the ECH 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.

[0065] The detection of the presence or absence of microorganisms in the ECH sample can be done by comparing the profile of the potential derivative or possibly the normalized profile of the potential difference derivative with the profile of the potential derivative or possibly the normalized profile of the potential difference derivative, 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.

[0066] 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.

[0067] For each compartment, we then obtain a results diagram similar to that of [Fig.3].

[0068] It should be noted that in each compartment C1-C10, after determining positivity in a compartment (i.e. the presence of bacteria in the sub-sample), identification is possible if the sub-sample contains only one bacterium. If the sub-sample contains, for example, two distinct types of bacteria, identification may still be possible if one of the bacteria grows faster than the other. In this case, it is indeed still possible to identify an electrochemical signature. In the case where the two bacteria grow at the same speed, identification will be difficult. Measuring device

[0069] As indicated above, the detection system comprises a measuring device suitable for implementing the method of the invention.

[0070] Two examples of embodiments of the measuring device are presented below.

[0071] In a first exemplary embodiment, the measuring device is in the form of a bottle 10 whose internal volume is compartmentalized. It comprises, for example, as many Cl-CIO compartments as there are fragments of the sample produced in the second step. Each Cl-CIO compartment is separated from an adjacent compartment. by a partition.

[0072] It should be noted that the device could have a greater number of compartments than there are sub-samples, for example to carry out measurements on reference samples.

[0073] Each compartment of the bottle 10 is instrumented so as to integrate at least two electrodes (measuring electrode E10 + reference electrode E20).

[0074] The bottle 10 may have means for dividing the ECH sample into several fragments. It should be noted that the different fragments obtained are not necessarily of identical volumes. The bottle 10 has, for example, a common inlet 11 leading to a distributor 12. The distributor 12 has several flow channels 13, each opening into a separate compartment.

[0075] The bottle 10 comprises, for example, on its external face, contact recovery electrodes, making it possible to connect each measuring electrode E10 and each reference electrode E20 to the measuring unit. On this point, it may be the principle already described in patent application WO2021 / 032654A1.

[0076] In a second exemplary embodiment, the measuring device comprises a well plate 100, each well 101 forming a separate compartment C1-C10 intended to receive a sub-sample S1-S10. Each well 101 is instrumented using at least one measuring electrode E10 and one reference electrode E20.

[0077] In this variant, the device comprises a controlled fluid distributor for delivering a fragment of the ECH sample into each well 101 of the plate 100.

[0078] In a non-limiting manner, the well plate 100 may consist of a first plate provided with through holes against which a second plate carrying the electrodes is applied and forming the bottom of each well.

[0079] In an identical manner, the measuring electrodes E10 and reference electrodes E20 associated with each compartment of the well plate are connected to the measuring unit via electrical connections.

[0080] It should be noted that the more measurements are carried out on a large number of fragments of the sample, the more sensitive the detection. In other words, the measuring device must have sufficient instrumented compartments, adapted to the volume of sample taken.

[0081] The invention thus presents numerous advantages, among which: - Reliable and sensitive detection, even in cases of polymicrobial infection; - Faster detection, enabled by fragmentation of the sample, this- increasing the microbial concentration in a fragment and also allowing the obtaining of negatives (sub-samples without bacteria); - A detection system equipped with a measuring device adapted to the implementation of the process; The use of an appropriate measuring device such as the compartmentalized bottle;

Claims

Claims

1. Method for detecting the presence of several microorganisms and for identifying said microorganisms in the same sample, characterized in that it comprises steps of: a. Fragmentation (E2) of the sample (ECH) into several sub-samples, b. Placement (E3) of each sub-sample (S1-S10) in a separate compartment (Cl-CIO), said compartments containing the same culture medium and each being instrumented using a measuring electrode (E10) and at least one reference electrode (E20), c. Measurement (E4) over time in each compartment of the potential difference between the measuring electrode (E10) and the reference electrode (E20), d. Determination of the profile of the derivative of the potential difference for each sub-sample (S1-S10), e. Detection (E5) of the presence or absence of each microorganism present in each subsample (S1-S10) and identification of the nature of each microorganism present, from the profile obtained in step d).

2. Method according to claim 1, characterized in that, for each sub-sample (S1-S10), the method 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 d).

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

4. Detection system, suitable for implementing the method as defined in one of the preceding claims, comprising a measuring device with several compartments (C1-C10), each compartment (C1-C10) being intended to receive a distinct sub-sample (S1-S10), characterized in that each compartment (C1-C10) is instrumented using at least one measuring electrode (E10) and one reference electrode (E20).

5. System according to claim 4, characterized in that the measuring device is in the form of a single bottle (10) whose volume internally comprises partitions arranged to form said compartments (Cl-CIO).

6. System according to claim 5, characterized in that the bottle (10) has a common inlet (11) and a distributor (12) having several flow channels (13) each opening into a separate compartment of the bottle.

7. System according to claim 4, characterized in that the measuring device comprises a plate (100) with wells, each well (101) forming a separate instrumented compartment.

8. System according to claim 7, characterized in that the well plate (100) comprises a first plate provided with through holes against which is applied a second plate carrying, for each through hole, a measuring electrode (E10) and a reference electrode (E20).

9. System according to one of claims 4 to 8, characterized in that each measuring electrode (E10) is produced in the form of a deposit of a conductive ink or an electro-deposition of a conductive measuring element.

10. System according to one of claims 4 to 9, characterized in that said reference electrode (E20) is produced in the form of a deposit of an Ag / AgCl ink covered with a polymer layer.

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