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 subsamples. 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 bloodstream 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 primarily consists of culturing blood in a nutrient broth under aerobic and anaerobic conditions, incubated at 36-37°C in automated systems adapted for this type of sample, in order to obtain bacterial growth. The objective is to increase the quantity of bacteria present in the sample by providing 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 because the bacterial concentration in the blood during bacteremia is always low, on the order of one or a few bacteria per milliliter of blood sampled. This first step of blood culture therefore consists of a simple detection of presence, without identification, from a sample without normal flora: any presence of bacteria or fungi thus leads to a positive test.

[0003] This detection will then be followed by culture on agar medium to isolate the pathogen in colony form, allowing for subsequent identification of the pathogen and its antibiogram. In the case of bloodstream infections, examinations are urgent, and the time to obtain results (positive blood culture, pathogen identification, antibiogram) has an undeniable impact on patient outcomes (mortality, length of hospital stay, complications, etc.). Blood culture results allow antibiotic therapy to be tailored to the patient's specific case: the earlier appropriate and effective antibiotic therapy is administered, 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 time it takes for blood cultures to become positive, as well as to reduce the analysis times for identification and antibiogram tests. Currently, two main types of automated analyzers 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 share the common feature of being implemented in automated systems, often large ones, with the impossibility of starting the test until the vial is in the system (pre-incubation can generate false negatives), hence the loss of precious hours before the implementation of optimized antibiotic therapy. A 2013 study showed that the average transport time was 9 hours (interquartile range: 3-15 hours), with 6% of vials having a transport time exceeding 20 hours.

[0006] Patent application EP4018191A1 describes the use of an instrumented container equipped with measuring electrodes, capable of holding a liquid sample for analysis. The detection of microorganisms is notably achievable by electrochemistry, by placing the container in a suitable chamber. This patent application proposes a simple solution for reducing the time between sample collection and the start of the analysis. This solution is also easily transportable, making it readily 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 generated electrochemical signatures only allow the identification of the species that multiplies the fastest, the colonization of the electrodes then preventing the appearance of a new potentiometric signature indicating the presence of a second species.

[0009] The object of the invention is to provide a method for making a sample more legible, particularly when the contamination is polymicrobial. Description of the invention

[0010] This objective is achieved by a method for detecting the presence of several microorganisms and identifying said microorganisms in the same sample, the method comprising the steps of: a. Fragmentation of the sample into several subsamples, b. Placement of each subsample in a separate compartment, said compartments containing the same culture medium and each being instrumented with 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 derivative profile of the potential difference for each subsample, 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 one particular feature, for each subsample, the process includes 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 characteristic, the said sample is blood.

[0013] The invention also relates to a detection system, adapted to the implementation of the process as defined above, comprising a multi-compartment measuring device, each compartment being intended to receive a separate sub-sample, each compartment being instrumented with 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 has partitions arranged to form said compartments.

[0015] According to one particular 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 one particular feature, the well plate comprises a first plate having 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 made 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 made in the form of a deposit of an Ag / AgCl ink covered with a polymer layer.

[0020] The microorganism is chosen for example 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 features and advantages will become apparent in the detailed description that follows, given in relation to the accompanying drawings, in which: - Fig. 1 illustrates the principle of implementation of the process of the invention; - Fig. 2 illustrates the principle of sample fragmentation; - Figure 3 illustrates the principle of sample fragmentation and allows us to highlight the detection of two bacteria A and B within the fragments of this sample; - Figures 4A and 4B show a first example of the realization of the measuring device of the invention, respectively seen from the side and from above of a cross-section; - Figures 5A and 5B show a second example of the 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 relates in particular to an easy-to-implement, fast and efficient method by which it is possible to detect but also to identify microorganisms contained in a liquid or solid sample using electrochemistry.

[0024] This process 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 the electrochemical signal, and in particular on the evolution of the derivative of the electrochemical potential with respect to time. With appropriate signal processing, it is possible to detect the growth of a microorganism, such as bacterial growth, and to guide identification.

[0026] The ECH sample may be a biological fluid, advantageously chosen 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, isolated bone marrow fluid, a cell extract, a tissue extract, an 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 recovered fluid, 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 beforehand and are not part of the invention.

[0027] The ECH sample can also be a liquid product from the food, pharmaceutical, or cosmetic industries. In a particular embodiment, the object from which the sample is taken can be chosen from large installations such as an industrial object, 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 equipment, surgical equipment...

[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 activities such as brushing teeth, dental care, or medical procedures involving probes or catheters. This is referred to as non-pathological bacteremia. Pathological bacteremia, on the other hand, corresponds to the presence of bacteria in the blood following pneumonia, a wound, or a urinary tract infection.

[0031] Among the microorganisms contained or likely to be contained in the sample, there are also 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] By way of illustrative and non-limiting examples of microorganisms contained or likely to be contained in the sample used in the context of the invention, one may mention 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 Pasteurella, bacteria of the genus Bacillus, bacteria of the genus Listeria, bacteria of the genus Clostridium, bacteria of the genus Mycobacteria, bacteria of the Enterococcus genus 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

[0033] In addition, among the bacteria of the order Enterobacterales contained or likely to be contained in the sample used in the context of the invention, we can mention the bacteria of the genera Escherichia, Salmonella, Shigella, Enterobacter, Klebsiella, Serratia, Proteus, Morganella, Yersinia, Citrobacter and Providencia.

[0034] With reference to [Fig.1], the process of the invention comprises the steps described below. First step El

[0035] The first step El consists of taking an ECH sample to be analyzed. As an example, 10mL 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 SI, S2, S3, S4, S5, S6, S7, S8, S9, S10.

[0037] Fragmentation of the ECH sample into several subsamples S1-S10 reduces detection time by increasing the microbial concentration in a fragment (through volume reduction). It also has the advantage of making the ECH sample more readable in the case of a polymicrobial infection.

[0038] Sample fragmentation also reduces detection time by recording negatives (bacteria-free subsamples) specific to the patient. This provides a personalized signal baseline, enabling more precise / faster detection.

[0039] Figure 2 illustrates the classic case of a blood culture. In this case, the bacterial load of the blood of a patient with bacteremia is on average 1 cfu / mL (cfu = colony-forming unit). For a blood culture, the sample is 10 mL. 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 in concentrations with two extreme situations: - The bacteria are distributed perfectly evenly, - The bacteria are all in the same compartment.

[0041] In both cases, the decrease 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 enough divisions, it is possible to separate the two species and thus bring the detection back to the classic case of a monomicrobial infection.

[0044] The number of fragments in the ECH sample is a parameter to be studied statistically in order 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 two cfu in the same subsample is very low (on the order of 1%). Using the Poisson distribution, we know that ten divisions are necessary (at a minimum) for a bacterial concentration of 1 cfu / mL in the test sample.

[0045] Figure 3 illustrates this principle in the case of a sample containing a first bacterium A (5 cfu) and a second bacterium B (5 cfu). This ECH sample is fragmented into ten subsamples S1-S10. Figure 3 shows an example of possible distributions of the two bacteria in the ten subsamples created. It can be seen that: - A subsample S6 contains only the A bacterium; - A subsample S1 contains only the B bacterium; - 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 subsample 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 subsamples S1-S10. The compartments are hermetically 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 subsample placed in the compartment, a measuring electrode E10 and a reference electrode E20.

[0050] 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 made by depositing a conductive ink or an electrode made by electro-depositing a conductive measuring element. Any conductive ink known to those skilled in the art can be used. In one particular embodiment, the conductive ink used is a metal ion-based ink or a conductive organic polymer 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", we mean 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 made 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 possibly identify the microorganisms contained in each subsample contained in each compartment.

[0054] The system also includes: - A measuring unit U1 of the potential difference between the measuring electrode E10 and the reference electrode E20, 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 power supply unit U4 to power the electronic acquisition unit U2 and possibly the processing unit U3.

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

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

[0057] The processing unit U3 may include a microprocessor and storage means. It may also include 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] By way of example, the U3 processing unit can be configured to: - Receive the potential difference measurement data from the acquisition unit, for example via the wireless link, - Establish an 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 by. - To order 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 related to the sampling, - Ensure the 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 (classical signature), a negative division (no detection) or a polymicrobial division (noisy signature). Fourth stage E4

[0060] This step E4 of the process 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 potential difference is measured continuously or discontinuously. In 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 it, 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 stage E5

[0063] This step is implemented by the U3 processing unit 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 more clearly observe the onset of 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 rate of potential change is observed, which is much more informative 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 subsample), identification is possible if the subsample contains only one bacterium. If the subsample 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 still possible to identify an electrochemical signature. If both bacteria grow at the same rate, identification will be difficult. Measuring device

[0069] As indicated above, the detection system includes a measuring device adapted to the implementation of the method of the invention.

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

[0071] In a first embodiment, the measuring device is in the form of a bottle 10 whose internal volume is compartmentalized. For example, it has as many Cl-ClO compartments as there are fragments of the sample prepared in the second step. Each Cl-ClO 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 subsamples, 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, for example, has contact electrodes on its outer face, allowing each measuring electrode E10 and each reference electrode E20 to be connected to the measuring unit. This may be the principle already described in patent application WO2021 / 032654A1.

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

[0077] In this variant, the device includes a controlled fluidic distributor to deliver a fragment of the ECH sample into each well 101 of the plate 100.

[0078] By way of non-limitation, the well plate 100 can consist of a first plate having through holes against which is applied a second plate carrying the electrodes and forming the bottom of each well.

[0079] Similarly, the measuring electrodes E10 and reference electrodes E20 associated with each compartment of the well plate are connected to the measuring unit via electrical links.

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

[0081] The invention thus offers numerous advantages, including: - Reliable and sensitive detection, even in the case of polymicrobial infection; Faster detection, made possible by fragmentation of the sample, which increases the microbial concentration in a fragment and also allows obtaining 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 a compartmentalized bottle;

Claims

Demands

1. A detection system adapted for implementing a method for detecting the presence of several microorganisms and identifying said microorganisms in the same sample, said method comprising the steps of: a. Fragmentation (E2) of the sample (ECH) into several sub-samples, b. Placement (E3) of each subsample (S1-S10) in a separate compartment (Cl-CIO), said compartments containing the same culture medium and each being instrumented with 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 derivative profile of the potential difference for each subsample (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.

3. Said system comprising a multi-compartment measuring device (C1-C10), each compartment (C1-C10) being intended to receive a separate subsample (S1-S10), characterized in that each compartment (C1-C10) is instrumented with at least one measuring electrode (E10) and one reference electrode (E20), the measuring device being in the form of a single bottle (10) whose internal volume has partitions arranged to form said compartments (C1-C10). System according to claim 1, 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. System according to claim 1 or 2, characterized in that each measuring electrode (E10) is made in the form of a deposit of a conductive ink or an electro-deposition of a conductive measuring element.

4. System according to any one of claims 1 to 3, characterized in that said reference electrode (E20) is made in the form of a deposit of an Ag / AgCl ink covered with a polymer layer.

5. System according to any one of claims 1 to 4, characterized in that said sample is blood.

Images