Method for determining the presence and / or degree of antimicrobial resistance of microorganisms

Abstract

The present invention proposes a method for determining the presence and / or degree of resistance of microorganisms to one or more antibacterial agents.

Description

Technical Field

[0001] The present invention belongs to the general field of microbiology, and more specifically, to the field of qualitatively / quantitatively determining the antibacterial susceptibility of microorganisms, particularly bacteria.

[0002] The present invention proposes a method for determining the presence and / or degree of resistance of a microorganism to one or more antibacterial agents. The present invention also relates to a more global method for analyzing a sample for the characteristics of a microorganism, including determining the presence and / or degree of resistance of the microorganism to one or more antibacterial agents by a method as described in the present invention.

Background Art

[0003] As a result of the repeated inappropriate use of antibiotics in the fields of medicine, food, biotechnology, the pharmaceutical industry, military and civilian defense, and environmental control, microorganisms resistant to these molecules have emerged. As a result of this inappropriate use, it has become increasingly difficult to fight infections caused by certain microorganisms, particularly certain bacteria.

[0004] Particularly in the field of treatment, when a microorganism is detected from a patient's biological sample, it is desirable to determine which antibiotics the microorganism is sensitive to. Currently, many bacterial species exist, and these bacterial species have become resistant to one or more classes of antibacterial agents. Therefore, in order to more accurately determine which antibiotics are optimal for administering to a patient, it has become more important to conduct susceptibility tests for antibiotics.

[0005] This is the case, for example, in the treatment of urinary tract infections (UTIs), where microbial analysis of the patient's urine is required.

[0006] UTI is a general term for infectious diseases at various sites of the urinary system.

[0007] Lower urinary tract symptoms include pain during urination, frequent urination, and the feeling of urinary urgency even when the bladder is empty. Symptoms of kidney infection usually include fever and flank pain in addition to the symptoms of UTI. In some cases, hematuria may also occur. In the very elderly and the young, symptoms may be vague or nonspecific. The common cause of infection is the presence of pathogenic E. coli in the urinary tract, but other bacteria, viruses, or fungi may also be the cause.

[0008] UTI is a common infectious disease and is usually treated with antibiotics, but it can cause severe complications such as renal failure. In order to avoid such complications and the overuse of antibiotics, efficient and targeted treatment is particularly important.

[0009] Methods for determining the susceptibility of microorganisms to antibiotics and common antibacterial agents have been proposed by the prior art. However, these methods are time-consuming, costly, and / or have low sensitivity, and none of them are completely satisfactory.

[0010] Therefore, there remains a need for a method for determining the presence and / or degree of resistance of microorganisms to antibacterial agents that is low-cost, highly sensitive, and can be implemented quickly. The present invention aims to provide such a method. In particular, the object of the present invention is to be able to implement this method without using expensive equipment, especially expensive optical equipment.

Summary of the Invention

[0011] The present invention proposes a method for determining the presence and / or degree of resistance of microorganisms to antibacterial agents using a universal label, namely a fluorescent compound, and a membrane having an upper surface with clearly defined different functions and a lower surface opposite thereto. In fact, the microorganisms adhere to the upper surface and express there, and the lower surface is in continuous contact with the culture solution, the fluorescent compound, and the washing solution. Another advantage of the present invention is that it does not require fixation to scan the microorganisms labeled with the fluorescent compound. The absence of this fixation allows the steps of labeling, washing, and scanning with the fluorescent compound to be repeated as many times as necessary.

[0012] Detection methods using fluorescent compounds are well known in the art, but as already mentioned, a fixation step is required and they are often used for microorganisms in the form of single cells to which the fluorescent compound is attached. In the present invention, microorganisms in the form of colonies and / or cell layers are placed on the upper surface of the membrane, and the fluorescent compound is brought into contact with the microorganisms through the lower surface of this membrane. It should be noted that prior to the present invention, it was not at all clear that a colony, which had been achieved with single cells, would function, that the fluorescent compound would diffuse uniformly during a short contact time, and that it would be sufficient for observation without the need for sophisticated and expensive equipment.

[0013] Furthermore, thanks to the labeling of the microorganisms and the washing through the lower surface of the membrane, there is no risk of removing the colonies of microorganisms and no fixation is required.

[0014] In the present application, all ranges must be interpreted as including their limits. The object of the present invention is to provide a method for determining the presence and / or degree of resistance of a microorganism to one or more antibacterial agents, each at one or more concentrations, the method comprising the following steps: a. forming a suspension of said microorganism in a liquid medium; b. uniformly attaching the microbial cells contained in said suspension to at least a part of the upper surface of the membrane; c. placing the lower surface of said membrane on a culture medium; d. placing one or more substrates each impregnated with one of said antibacterial agents in contact with said membrane, preferably placing said one or more substrates each impregnated with one of said antibacterial agents on the upper surface of said membrane to which the microbial cells are attached; e. incubating the whole under conditions suitable for promoting the growth of said microorganism; f. contacting the lower surface of said membrane with a fluorescent compound capable of labeling an endogenous component of said microorganism in order to label said microbial cells; g. optionally, washing the lower surface of said membrane to remove excess fluorescent compound; h. Detecting the presence or absence of fluorescence around the substrate on the upper surface of the film using a fluorescence reading optical device; i. Determining the presence and / or degree of resistance of the microorganism to the antibacterial agent based on the detection of the presence and / or size of a non-fluorescent region around the substrate.

[0015] "Resistance" means the ability of a microorganism to resist the effect of an antibacterial agent.

[0016] "Microorganism" means a microscopic organism, particularly bacteria, viruses, or fungi.

[0017] The microorganisms whose susceptibility to one or more antibacterial agents is tested according to the method of the present invention are eukaryotic organisms and particularly unicellular organisms, or prokaryotic organisms and particularly unicellular organisms.

[0018] The eukaryotic microorganism may be a yeast of the genus Saccharomyces or a yeast of the genus Candida such as Candida albicans, a fungus, an alga, or a mixture thereof.

[0019] The prokaryotic microorganism is a bacterium, which may be Gram-positive or Gram-negative, or an archaebacterium or a mixture thereof.

[0020] Therefore, the microorganisms whose susceptibility to one or more antibacterial agents is tested according to the method of the present invention are preferably selected from yeasts, fungi, algae, Gram-positive bacteria, Gram-negative bacteria, archaebacteria, and mixtures thereof.

[0021] Among bacteria, by way of example and in a non-exhaustive manner, bacteria belonging to the branches of Spirochetes and Chlamydia, the family Alcaligenaceae, the family Pasteurellaceae, the family Enterobacteriaceae, the genus Enterococcus, the genus Staphylococcus, the family Pseudomonadaceae, the genus Streptococcus, the genus Micrococcus, the family Legionellaceae, the genus Mycobacterium, the family Bacillaceae and the genus Cyanobacteria can be mentioned. More specifically, these bacteria are selected from the group consisting of Bordetella pertussis, Haemophilus influenza, Citrobacter freundii, Escherichia coli, Enterococcus faecalis, Klebsiella pneumoniae, Klebsiella aerogenes, Legionella pneumophila, Morganella morganii, Pseudomonas aeruginosa, Proteus mirabilis, Serratia marcescens, Bacillus anthracis, Staphylococcus aureus, Staphylococcus saprophyticus, and Streptococcus agalactiae.

[0022] Preferably, the microorganism is Escherichia coli, Pseudomonas, Candida and / or Staphylococcus.

[0023] Among archaea, by way of example and in a non-exhaustive manner, archaea belonging to the phyla Crenarchaeotes and Euryarchaeotes can be mentioned.

[0024] Of course, the method of the present invention can also be applied to a mixture of microorganisms. Therefore, in this specification, the term "microorganism" includes a mixture of microorganisms, and the global sensitivity to the antibacterial agent thereof is evaluated by the method of the present invention.

[0025] The "antibacterial agent" means a natural or synthetic substance that kills or inhibits the growth of microorganisms such as bacteria, fungi, and algae.

[0026] Examples of antibacterial agents include, but are not limited to, antibacterial agents, antifungal agents, antiviral agents, antiparasitic agents, broad-spectrum therapeutic agents, non-pharmaceutical antibacterial agents (such as essential oils and antibacterial insecticides), ozone, antibacterial scrubs, halogens, alcohols, phenols, and phenolic compounds.

[0027] Preferably, the antibacterial agent according to the present invention has antibacterial properties.

[0028] In a preferred embodiment of the present invention, at least one, preferably all, of the antibacterial agents are antibiotics.

[0029] In step (a) according to the present invention, a suspension of microorganisms in a liquid medium is formed.

[0030] Therefore, according to the present invention, any suitable method can be implemented. As an example, the microorganisms can be transferred from the original medium to a liquid medium.

[0031] The "original medium" means any sample that may contain microorganisms due to its properties after contamination, whether liquid or solid.

[0032] Preferably, the original medium according to the present invention is a liquid sample.

[0033] Typically, this liquid sample can be a biological fluid such as plant fluids like sap, nectar, and exudates from roots; samples in a culture medium such as cell cultures of higher eukaryotes, yeast, fungi, or algae, or in a biological culture reactor; liquids obtained from animal or plant cells; liquid samples obtained from animal or plant tissues; liquid samples obtained from a food matrix; liquid samples from a chemical reactor; water from a municipality, river, pond, lake, sea, or an air-cooled tower; samples from liquid industrial waste; wastewater, especially from intensive animal production or industries in the chemical, pharmaceutical, cosmetic, and nuclear fields; liquid samples from pharmaceuticals; liquid samples from cosmetics; fragrances; soil samples, or mixtures thereof.

[0034] Within the scope of the present invention, the sample can be obtained by any type of sampling, such as contact, scraping, perforation, extraction, washing, rinsing, suction, pumping, etc.

[0035] When the liquid sample is a biological fluid, the latter is preferably selected from the group consisting of blood such as whole blood or anticoagulated whole blood, serum, plasma, lymph, saliva, sputum, tears, sweat, sperm, urine, feces, milk, cerebrospinal fluid, interstitial fluid, fluid isolated from bone marrow, mucus or fluid isolated from the respiratory tract, intestinal tract, or urogenital tract, cell extracts, tissue extracts, and organ extracts. Thus, the biological fluid can be any fluid naturally secreted or excreted from the human or animal body, or any fluid recovered from the human or animal body by any technique known to those skilled in the art, such as extraction, sampling, washing, etc. The steps of recovering and isolating these different liquids from the human or animal body are performed before implementing the method according to the present invention. Preferably, the liquid sample is urine.

[0036] Similarly, if one of the samples envisaged, for example, due to its particularly solid nature, its concentration, or the presence of solid residues, waste, suspensions, or interfering molecules, cannot be processed by the method according to the present invention, the sampling as defined below further includes a preliminary step of preparing the material while optionally solubilizing the sample by techniques known to those skilled in the art, such as filtration, precipitation, dilution, distillation, mixing, concentration, dissolution, etc.

[0037] For example, when the liquid sample is urine, the urine can be filtered to remove interfering substances such as crystals, hyaline structures, cell aggregates and / or large eukaryotic cells. For this purpose, a sterile sieve device with a porosity of 5 μm or 10 μm such as a pluriStrainer can be used. However, this filtration is optional.

[0038] Preferably, the suspension of microorganisms in the liquid medium formed according to the present invention is prepared from isolated colonies.

[0039] Preferably, the suspension of microorganisms in the liquid medium formed according to the present invention has a McFarland value between 0.4 and 0.6.

[0040] The "McFarland value" means a standard for standardizing the approximate number of bacteria in a liquid suspension by comparing the turbidity of the test suspension with the turbidity of the McFarland standard. Since the turbidity of a microbial suspension is proportional to the number of microbial cells in the suspension, the number of cells in the suspension is within a relevant range for standardizing microbiological tests.

[0041] Preferably, the McFarland value of the suspension in step (a) according to the present invention is about 0.5.

[0042] In a particular embodiment of the present invention, the liquid medium is a nutrient medium for microorganisms such as liquid lysogeny broth (LB) medium, Mueller-Hinton broth (MH) medium, or physiological saline (0.9% NaCl), and the medium is seeded with microorganisms and cultured under conditions that promote the growth of the microorganisms until a sufficient number of cells are reached to obtain the desired McFarland value.

[0043] Any other means of forming a microbial suspension with a McFarland value of 0.4 - 0.6 is also included within the scope of the present invention, for example, dilution of an overly concentrated cell suspension already available from a stage prior to detecting and / or identifying the microorganisms in the original sample.

[0044] The term "membrane" refers to a thin wall of a porous substance that is interposed between two media and enables the removal or concentration of specific components.

[0045] It is clear that the membrane implemented in the present invention can retain the microorganisms contained in the suspension and / or is impermeable to these microorganisms. Advantageously, this membrane is porous with naturally occurring pores or artificially created pores and has a porosity suitable for retaining the microorganisms present in the suspension. Advantageously, this porosity is 0.5 μm or less, particularly 0.45 μm or less.

[0046] Any membrane that is usually implemented to filter a liquid sample and retain the microorganisms present therein can be used within the present invention. Membranes suitable for use in the present invention include, but are not limited to, membranes made of polytetrafluoroethylene (PTFE), polyester, polycarbonate, nylon, polyvinylidene fluoride (PVDF), cellulose and cellulose derivatives such as cellulose acetate, cellulose nitrate, regenerated cellulose, nitrocellulose, cellophane, or mixed cellulose ester (MCE). Such membranes are particularly available from Whatman, Merck Millipore, and Sigma Aldrich.

[0047] Advantageously, the membrane implemented in the present invention is an MCE membrane, particularly a black MCE membrane, and more specifically, the black MCE membrane No. 1 sold by Merck Millipore.

[0048] It should be noted that the membrane implemented in the present invention does not necessarily need to be pretreated before step (b). This pretreatment may be a coating, particularly a coating with a microorganism-binding molecule as defined in [1]. Preferably, the membrane implemented in the present invention is not pretreated before step (b).

[0049] Step (b) of the method according to the present invention involves homogeneously attaching the microbial cells contained in the suspension to at least a part of the upper surface of the membrane. In this step, the membrane may or may not have its lower surface already placed on the culture medium.

[0050] Such an arrangement step can be carried out, for example, by filtering at least a part of the suspension on the membrane and collecting the microbial cells there. In such an embodiment, it is preferable that the membrane is not yet placed on the culture solution. Otherwise, for example, a small amount of the suspension can be placed on the membrane and the placed cells can be homogeneously spread thereon using beads or any other conventional means. In such an embodiment, it is preferable that the membrane is already on the culture medium.

[0051] The "culture medium" means a substance containing nutrients capable of supporting the growth of microorganisms.

[0052] In a first embodiment, the culture medium implemented in step (c) of the method according to the present invention is a liquid culture medium.

[0053] This liquid culture medium implemented in step (c) of the method according to the present invention may be any liquid culture medium suitable for culturing microorganisms known to those skilled in the art. The latter has a carbon source such as glucose or glycerol; a nitrogen source such as ammonium or nitrate 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.

[0054] Examples of liquid media suitable for use in step (c) of the method according to the present invention include Nutrient Broth No. 1 (NB1), Nutrient Broth No. 2 (NB2), Mueller-Hinton Broth, Lysogenic Broth (LB) (also known as Luria-Bertani), Tryptic Soybean (TSB) culture medium, and Brain Heart Infusion (BHI) broth. Such media are available, in particular, from ThermoFisher, Bio-Rad Laboratories, and Sigma Aldrich.

[0055] These different liquid media can be supplemented by one or more elements selected from the group consisting of antioxidants such as sodium pyruvate, glutamic acid, calcium chloride, and magnesium chloride; multivitamins such as Polyvitex (PVX); iron sources such as ferric citrate or Vitox®; protein supplements such as bovine serum albumin (BSA); and anticoagulants such as citrate.

[0056] Advantageously, the liquid medium is sterile or is sterilized before contacting the lower surface of the membrane in step (c) of the method according to the present invention. The culture medium can be sterilized by various techniques known in the art, such as autoclaving and / or the use of aseptic techniques for preparing the culture medium, but is not limited thereto.

[0057] In a second embodiment, the medium used in step (c) of the method according to the present invention is a solid medium.

[0058] As a variant of this second embodiment, the solid medium may be a nutrient gel. The latter may be any nutrient gel suitable for the culture of microorganisms known to those skilled in the art. With respect to the liquid medium, this nutrient gel has a carbon source such as glucose or glycerol; a nitrogen source such as ammonium or nitrate or amino acids; and salts and / or trace elements and vitamins for the growth of microorganisms. In particular, this solid medium may not be selective for a particular type of microorganism. Alternatively, it may be selective for a particular type of microorganism.

[0059] In this second embodiment, the solid medium may be a diffusion intermediate impregnated with the liquid medium as previously defined. The diffusion intermediate is a solid support that must be porous in order to allow good impregnation with the liquid medium as previously defined. Advantageously, the diffusion intermediate is, in particular, a pad of a material selected from the group consisting of paper of cellulose nature; cotton paper; agarose; gelatin; cellulose; methylcellulose; carboxymethylcellulose; nitrocellulose; cellulose acetate ester; alginic acid; polyolefin; a porous solid support such as a porous membrane, in particular an ion-exchange porous membrane; a membrane of a perfluorinated polymer such as a Sephadex-type resin or PVDF packaged as a membrane; a felt fabric; a glass fiber fabric; a membrane of an organic polymer such as polyethylene, polypropylene or a mixture thereof; a nylon fabric; a polyacrylamide gel; a Sepharose gel and mixtures thereof.

[0060] Examples of solid media suitable for use in step (c) of the method according to the present invention include nutrient agar, tryptic soy agar (TSA), Mueller-Hinton agar, Legionella glycine vancomycin polymyxin cycloheximide (GVPC) agar, buffered charcoal yeast extract (BCYE) agar, Buffered cefaMandole Polymyxine Anisomycine α-cetoglutarate (BMPAα) agar medium, Reasoner's 2A (R2A) agar medium, Sabouraud Dextrose Agar (SDA), Columbia agar medium with horse blood, blood agar media such as sheep blood agar medium, chocolate agar medium with Vitox, Haemophilus Test Medium (HTM) agar medium, charcoal agar medium, and the like. Such media are particularly available from ThermoFisher, Oxoid, and VWR.

[0061] Advantageously, when the medium carried out in step (c) of the present invention is solid, it is blood agar.

[0062] Furthermore, when using a solid medium, it is also possible to add a liquid medium as defined above between the solid medium and the membrane on which the microorganism is present on the upper surface. However, this addition is optional.

[0063] Step (d) of the method according to the present invention consists of placing one or more substrates each impregnated with one of the antibacterial agents on a part of the upper surface of the membrane on which the microbial cells are placed.

[0064] The "substrate" means any support that can at least partially absorb the antibacterial agent and diffuse it into its environment.

[0065] In one embodiment, step d is performed before step b or before step c. In this case, the antibacterial agent diffuses from the substrate through the membrane to the membrane surface. "Before step b" preferably means that step d is carried out between steps a and b. In that case, step d also includes the sub-step of "placing one or more substrates each impregnated with one of said antibacterial agents in contact with said membrane" before "providing the membrane". "Before step c" preferably means that step d is carried out between steps b and c.

[0066] In other embodiments, step d is performed after step c. In this case, the antibacterial agent diffuses from the substrate to the upper surface of the membrane and forms an antibacterial gradient there. This alternative embodiment is preferred.

[0067] During the subsequent step (e) of the method of the present invention, the inhibition of the growth of microorganisms on the membrane around the substrate indicates the susceptibility of this microorganism to this antibacterial agent. The larger the growth inhibition area, the lower the degree of resistance of this microorganism to the antibacterial agent. If there is no growth inhibition area on the membrane, it indicates the resistance of the microorganism to the tested antibacterial agent.

[0068] For the method according to the present invention, a single substrate can be used to evaluate the susceptibility of a microorganism to a single antibacterial agent impregnated in the substrate. Otherwise, a plurality of substrates each impregnated with a different antibacterial agent can be used to evaluate the susceptibility of the microorganism to said plurality of antibacterial agents. In such an embodiment of the present invention, the substrates are preferably arranged on the membrane so as to be well separated from each other so as not to interfere with each other, as recommended by laboratory test guidelines.

[0069] The substrate implemented in accordance with the present invention is a disk and / or an elongated strip impregnated with a gradient of an antibiotic. Preferably, the substrate implemented in accordance with the present invention is either a disk impregnated with a gradient of an antibiotic or an elongated strip (Etest® strip). In particular, in these embodiments, the method according to the present invention makes it possible to determine the presence and the degree of resistance of microorganisms to one or more antibiotics.

[0070] In a particular embodiment of the method according to the present invention, at least one substrate, preferably all substrates, are disks each impregnated with a single concentration of an antibacterial agent.

[0071] In an embodiment of the method according to the present invention, at least one substrate, preferably all substrates, are strips impregnated with a concentration gradient of an antibacterial agent along their length.

[0072] Advantageously, the substrate implemented in accordance with the present invention is made of paper or plastic. Preferably, it is made of plastic.

[0073] Preferably, the substrate implemented in accordance with the present invention is a strip impregnated with a gradient of an antibiotic, such as sold under the trade name Etest®. The strip is made of a thin, inert and non-porous plastic slide having a dilution range according to a predefined antibiotic gradient on one side and a MIC measurement scale in μg / mL units on the other side.

[0074] Step (e) of the method according to the invention comprises incubating the whole under conditions suitable for promoting the growth of said microorganism, i.e., the microorganism is maintained in an environment where it can increase in number by cell division to form microcolonies, colonies, or a layer of cells. This environment includes not only the culture medium brought into contact with the lower surface of the membrane, but also conditions such as temperature and atmosphere that enable cell growth. Typically, the temperature implemented in step (e) is composed of between 25 °C and 40 °C. Specific examples of the temperature implemented in step (e) are 30 °C, 32 °C, 37 °C, and 39 °C. Usually, regarding the atmospheric conditions, step (e) can be implemented under air, under air + 5% CO2, or under a mixed gas consisting of 5% O2, 5% CO2, and 90% N2.

[0075] Typically, depending on the type of microorganism present in the liquid sample, step (e) must be 8 hours or less, particularly 7 hours or less, particularly 6 hours or less, and even more particularly 5 hours or less. In particular, step (e) must be carried out for at least 2 hours, particularly at least 2.5 hours, particularly at least 3 hours. In fact, for example, from the results obtained by the inventors, particularly under the conditions disclosed in the following Examples section, colonies of Escherichia coli or Staphylococcus aureus can be detected 4 hours after culturing with the culture medium.

[0076] In a particularly advantageous embodiment of the invention, the suspension formed in step (a) has a McFarland value between 0.4 and 0.6, and the culture step (e) is carried out for between 3 hours and 6 hours.

[0077] Step (f) of the method according to the invention is a labeling step for labeling the microbial cells grown on the upper surface of the membrane obtained after steps (b) to (e) with a fluorescent compound.

[0078] Generally, the term "fluorescent compound" refers to a compound that emits light when excited by light of another appropriate wavelength. The term "fluorescence" as applied to a compound can be used to refer to the property of absorbing energy (such as UV, visible, or IR radiation) and re-emitting at least a portion of that energy as light over time.

[0079] The present invention implements a fluorescent compound that can recognize and bind to an endogenous component of a microorganism. By definition, an endogenous component means a component that is derived within the microorganism.

[0080] Typically, the fluorescent compound implemented in step (f) of the method according to the present invention can recognize, bind to, and thus label mitochondria, cell membranes, or nucleic acids. Alternatively, the fluorescent compound implemented in step (f) of the method according to the present invention can also label the cytoplasm.

[0081] In a first embodiment, the fluorescent compound implemented in step (f) of the method according to the present invention can recognize, bind to, and thus label mitochondria. Advantageously, this fluorescent compound is selected from the group consisting of Rhodamine 123, DiOC6(3), DiOC7(3), JC-1, MitoTracker Orange, and Red CMXRos.

[0082] In a second embodiment, the fluorescent compound implemented in step (f) of the method according to the present invention can recognize, bind to, and thus label cell membranes. Advantageously, this fluorescent compound is selected from the group consisting of FM1-43 and FM4-64.

[0083] In a third embodiment, the fluorescent compound implemented in step (f) of the method according to the present invention can recognize, bind to, and thus label nucleic acids. "Nucleic acids" is understood to mean ribonucleic acid (RNA) such as single-stranded or double-stranded deoxyribonucleic acid (DNA), messenger RNA, or ribosomal RNA.

[0084] Fluorescent compounds specific to nucleic acids are selected particularly from fluorescent intercalating agents, dyes that bind to base A:T or base G:C, and permeable or non-permeable cyanines. More specifically, the fluorescent compounds are selected from the group consisting of ethidium bromide, thiazole orange, thiazole blue and their derivatives, thioflavin S, thioflavin T, thioflavin TCN, diethylquinolylthiocyanine iodide (DEQTC), TOTO1, TOPRO1, TOTO3, TOPRO3, YOYO1, Hoechst 33258, Hoechst 33342, Hoechst 34580, 4',6-diamidino-2-phenylindole (DAPI), pyronin Y, acridine orange, oramine O, calcein, oramine O, oxazine 750, astral blue, and particularly the SYTO series including SYTO 11, SYTO 12, SYTO 13, SYTO 15, SYTO 16, SYTO 18, SYTO 62, SYTO 80 or SYTO 81.

[0085] The fluorescent compound can be selected from the group consisting of 4',6-diamidino-2-phenylindole (DAPI), Rhodamine123, DiOC6(3), DiOC7(3), JC-1, MitoTracker Orange, Red CMXRos, FM1-43, FM4-64, thiazole orange, thiazole blue and its derivatives, TOTO1, TOPRO1, TOTO3, TOPRO3, YOYO1, Hoechst 33258, Hoechst 33342, Hoechst 34580, acridine orange, calcein, oramine-O, oxazine 750, astral blue, and particularly the SYTO series including SYTO 11, SYTO 12, SYTO 13, SYTO 15, SYTO 16, SYTO 18, SYTO 62, SYTO 80 or SYTO 81.

[0086] In a specific embodiment, the fluorescent compound implemented in step (f) of the method according to the present invention is 4',6-diamidino-2-phenylindole (DAPI).

[0087] In the fourth embodiment, the fluorescent compound implemented in step (f) of the method according to the present invention can label the cytoplasm.

[0088] In a specific embodiment of this fourth embodiment, the fluorescent compound implemented in step (f) can be released from a molecular probe that itself disintegrates in response to a stimulus such as the presence of an enzyme, releasing the fluorescent compound. For example, to label the cytoplasm, the molecular probe may be 5(6)-CFDA (5-(and-6)-Carboxyfluorescein Diacetate) or the SmartID Green Probe for Alanine aminopeptidase commercially available from Molsid.

[0089] In this step (f), the fluorescent compound is brought into contact with the lower surface of the membrane and permeates the membrane.

[0090] In a specific embodiment, the lower surface of the membrane can be brought into contact with a solution or dispersion of the fluorescent compound. The fluorescent compound is preferably present in excess in the solution or dispersion relative to the microorganisms present on the upper surface of the membrane.

[0091] In another specific embodiment, the lower surface of the membrane can be brought into contact with a solution or dispersion of the fluorescent compound as defined above via a diffusion intermediate impregnated with the solution or dispersion. The diffusion intermediate is as defined above for the solid medium, provided that in step (e) this diffusion intermediate must be a porous solid support to allow good impregnation with the solution or dispersion of the fluorescent compound. A specific example of such a diffusion intermediate useful in step (f) is a glass fiber cloth impregnated with a solution or dispersion of a fluorescent compound such as a DAPI solution, more particularly a solution containing 1.4 μg / ml of DAPI.

[0092] Typically, the duration of step (f) of the method according to the present invention is 30 minutes or less, preferably 25 minutes or less, particularly 20 minutes or less, especially 15 minutes or less, and more particularly 10 minutes (i.e., 10 minutes ± 2 minutes).

[0093] Step (f) of the method according to the invention can be carried out at a temperature between 10°C and 40°C, preferably between 15°C and 30°C, more particularly at room temperature (i.e. 23°C ± 5°C).

[0094] In particular, step (f) of the method according to the invention can be carried out in the dark.

[0095] Step (g) of the method according to the invention is an optional washing step and can remove the fluorescent compound present on the lower surface of the membrane. In fact, the presence of this fluorescent compound adsorbed on the lower surface of the membrane may generate background noise during step (h).

[0096] The lower surface of the membrane is subjected to at least one rinse to remove all traces of the fluorescent compound and / or the solution or dispersion containing it. This rinse is usually carried out using a rinse solution, and in particular, an aqueous rinse solution that does not affect the microbial cells on the upper surface of the membrane is used. This aqueous solution also serves to eliminate non-specific interactions. This aqueous solution can consist of one or more of the following components: buffers such as Tris, phosphate, acetate, borate buffers; salts such as KCl, NaCl, (NH4)2SO4, MgCl2, CaCl2; and detergents or surfactants such as Tween®, Triton®, sodium dodecyl sulfate (or SDS), bovine serum albumin (BSA). This rinse can be repeated 2, 3, 5, 10, or even 50 times, using the same or different rinse solutions for each rinse. Typically, rinsing is repeated 3 times using a solution consisting of phosphate buffered saline (or PBS) and 0.1% Tween® with a pH of 7 - 7.5.

[0097] Advantageously, this washing step (g) can be carried out by continuously flowing the rinse solution at the level of the lower surface of the membrane. This can be carried out using a membrane washing device well known in the art, such as the washing module sold by DIAMIDEX.

[0098] Typically, the duration of step (g) of the method according to the invention is 40 minutes or less, preferably 30 minutes or less, particularly 20 minutes or less, especially 15 minutes (i.e., 15 minutes ± 3 minutes).

[0099] Step (g) of the method according to the invention can be carried out between 10 °C and 40 °C, preferably between 15 °C and 30 °C, particularly at room temperature (i.e., 23 °C ± 5 °C).

[0100] In particular, step (g) of the method according to the invention is carried out in the dark.

[0101] Step (h) of the method according to the invention consists of placing the membrane obtained after step (g) under a microscope stage that can be scanned by an optical device capable of reading fluorescence, for example, by an excitation source adapted to the fluorescent compound used.

[0102] In one embodiment of the method carried out according to the invention, an image of the membrane is captured at one or more intervals using at least one image capture device.

[0103] Usually, the excitation source excites the fluorescent compound that labels the microorganism and subsequently measures the emission of fluorescence from this compound.

[0104] For the positioning of the substrate in step (d), the membrane itself can be manually manipulated by moving the membrane or by moving the optical device on which the membrane is placed. Alternatively, the optical device can be automatically controlled to scan the membrane on the stage.

[0105] Light sources capable of emitting in the ultraviolet, visible and / or near-infrared spectra, which are well-known to those skilled in the art, can be used in the present invention. For example, the light source may be a continuous lamp such as a deuterium or xenon arc lamp for generating ultraviolet light, or a tungsten halogen lamp for generating visible / near-infrared excitation. Alternatively, the light source may be one or more LEDs. These light sources provide a wide emission range, and the spectral bandwidth for a specific excitation wavelength can be reduced using filters such as optical interference filters, prisms and / or diffraction gratings.

[0106] Therefore, in a specific embodiment of the present invention, the light source in the optical device is composed of one or more LEDs (plural available).

[0107] Those skilled in the art will know which excitation light source should be used according to the fluorescent compound implemented in the method according to the present invention. Advantageously, the excitation source implemented in step (h) of the method according to the present invention is an optical microscope equipped with a filter such as a UV filter or a Cy5 filter.

[0108] The measurement data obtained during the execution of step (h) can be analyzed by an algorithm and compared particularly with the control measurement values.

[0109] Typically, the duration of step (h) of the method according to the present invention is 20 minutes or less, advantageously 15 minutes or less, particularly 10 minutes or less, particularly 7 minutes (i.e., 7 minutes ± 2 minutes).

[0110] Considering the durations of the different steps of the method according to the present invention, the latter steps (b) to (i) are carried out in less than 10 hours, particularly less than 9 hours, preferably less than 8 hours, more preferably less than 7 hours, even more preferably less than 6 hours, and even more preferably less than 5 hours.

[0111] The optical device involved in step (h) of the present invention is a simple and inexpensive optical device.

[0112] In one embodiment, the optical device in step (h) has a magnification between 1 and 50 and an optical resolution composed between 6 and 20 Mpix.

[0113] Preferably, this optical device has a magnification between 1 and 8, more preferably between 1 and 4, for example between 2 and 4.

[0114] Preferably, the optical resolution of the optical device is composed of 9 to 15, more preferably 11 to 13 Mpix.

[0115] The presence and / or degree of resistance of said microorganism(s) to said antibacterial agent(s) is determined in step (i) of the method of the invention based on the absence or presence of a non-fluorescent region detected around said substrate(s) and optionally on the size. Determining the degree of resistance of a microorganism to an antibacterial agent based on the size of the growth inhibition zone is within the skill of the art.

[0116] In particular, for all types of microorganisms, the present invention makes it possible to determine the presence and / or degree of resistance of said microorganism(s) to said antibacterial agent(s) by shortening the duration of the method by 4 to 5 times compared to the gold standard (Etest on agar).

[0117] More specifically, when using an elongated strip impregnated with a gradient of an antibiotic, the method according to the invention can determine the presence and the degree of resistance of the microorganism to said antibiotic by simply reading the image taken by the optical device (see Figure 3).

[0118] In one embodiment, the method according to the invention includes determining the minimum inhibitory concentration (MIC) of an antibacterial agent against a microorganism.

[0119] The "minimum inhibitory concentration (MIC)" means the lowest concentration of a chemical substance, usually a drug, that inhibits the visible growth of the microorganism of interest.

[0120] The MIC varies depending on the microorganism and the antimicrobial agent itself. The MIC is often expressed in micrograms per milliliter (μg / mL) or milligrams per liter (mg / L).

[0121] Advantageously, in the method of the present invention, the MIC is evaluated using a strip comprising a predefined continuous gradient of antibiotic concentration, such as, for example, E-test® or the like.

[0122] Preferably, the strip is graduated and indicates the concentration of the antibiotic present at all heights of the strip, facilitating the interpretation of the results.

[0123] To obtain an inhibition ellipse for determining the MIC, according to the present invention, the strip is simply placed on the seeded membrane and incubated. After the incubation period, the MIC value (μg / mL) can be read directly on the scale at the intersection of the inhibition ellipse and the strip. This MIC value serves as an indicator for those skilled in the art to select the antimicrobial agent and its amount for optimal treatment. In fact, the MIC value read on the strip is compared with the critical concentration defined by the pathogen / antibiotic pair (EUCAST: European Committee on Antimicrobial Susceptibility Testing). If the MIC value read on the strip is lower than the minimum critical concentration, the pathogen is said to be sensitive to the antibiotic at the standard dose, and if the MIC value read on the strip is higher than the maximum critical concentration, the pathogen is said to be resistant to the antibiotic. Finally, if the MIC value read on the strip is between the minimum and maximum critical concentrations, the pathogen is said to be intermediate or sensitive to the antibiotic at a high dose.

[0124] Another aspect of the present invention relates to a method for analyzing a sample for its microbial characteristics, the method comprising: - detecting the presence of at least one microorganism in the sample, - optionally, identifying the microorganism, - Recovering the microorganism from the sample, - Implementing the method according to the invention, which determines, for the recovered microorganism, the presence and / or degree of resistance of the microorganism to one or more antimicrobial agents at one or more concentrations.

[0125] The step of detecting the presence of at least one microorganism in the sample and optionally identifying the microorganism can be carried out by any method known in the art.

[0126] The sample can be any of a biological fluid; a plant fluid such as sap, nectar, and exudates from roots; a sample in a culture medium such as a cell culture of higher eukaryotes, yeast, fungi, algae, or in a biological culture reactor; a liquid obtained from one (or more) animal or plant cells; a liquid sample obtained from animal or plant tissue; a liquid sample obtained from a food matrix; a liquid sample from a chemical reactor; municipal water, river water, pond water, lake water, seawater, or water from an air-cooled tower; a sample from liquid industrial waste, particularly wastewater from intensive animal production or industries in the chemical, pharmaceutical, cosmetic, and nuclear fields; a liquid sample from a pharmaceutical product; a liquid sample from a cosmetic product; a fragrance; a soil sample, or a mixture thereof. It can correspond to any of the above characteristics regarding the original medium containing the microorganism to be assayed.

[0127] Other features and advantages of the present invention will be more apparent to those skilled in the art from the following examples, given by way of illustration and not limitation, with reference to the accompanying drawings.

Brief Description of the Drawings

[0128]

Figure 1

Figure 2

Figure 3

Mode for Carrying Out the Invention

[0129] [Use of the Method According to the Invention for the Evaluation of MIC (Minimum Inhibitory Concentration)] A. Materials and Methods (Samples) The following strains were used in this study: - Escherichia coli (gentamicin-sensitive) - DSM1103 - Escherichia coli (gentamicin-resistant) - DSM1103 ATCC BAA-2452 - Staphylococcus aureus (oxacillin-sensitive) - DSMZ 2568 - Staphylococcus aureus (oxacillin-resistant) - DSMZ 28766

[0130] Description of the resources and equipment used (Equipment) The equipment to be used includes a filter bench, a rinse bench, and an optical bench. The filter bench operates with a vacuum pump and can filter 6 samples in parallel. The rinse bench is continuously operated by a pump and enables simultaneous washing of 6 specimens. This washing is performed in series, and the washing liquid flows between the 6 samples and is recycled. The optical bench is equipped with LEDs and filters and can read blue fluorescence (DAPI channel) or red fluorescence (FM4-64 channel).

[0131] (Impregnated substrate) The substrate used is a BioMerieux Etest® strip impregnated with gentamicin (for experiments with Escherichia coli) or oxacillin (for experiments with Staphylococcus aureus), respectively. The references for the E-tests used are as follows: - ETEST gentamicin: 412368 - ETEST oxacillin (= methicillin): 412432

[0132] Protocol J-1: Preparation of bacterial samples - Inoculate different strains on TSA plates by the streak method (1 strain per plate), - Incubate at 37 °C for 24 hours.

[0133] J0: AST: Filtration or no filtration, and incubation · Protocol without a filtration step - Collect several colonies of the same size and morphology with 2 mL of physiological saline (0.9% NaCl), - Adjust the McFarland value to 0.5 with a nephelometer, - Place a 45 mm black MCE membrane with a porosity of 0.45 μm (HABG047S6, manufactured by Millipore) on an MH agar medium pre-incubated at 37°C, ensuring no bubbles form. - Apply 1 mL of a suspension with a McFarland value of 0.5 of the corresponding strain to the MCE membrane. - Add 10 sterilized glass beads with a diameter of 2 mm, and rotate the box 10 times up and down, then 10 times from left to right, and finally 10 times clockwise and 10 times counterclockwise to shake. - Place the E-test with plastic forceps so that no bubbles form, ensuring good diffusion of the antibiotic in the agar. - Incubate at 37°C for 3, 4, or 6 hours with the E-test agar medium facing upwards.

[0134] · Protocol with an alternative filtration step (not performed in this case) - Collect several colonies of the same size and morphology with 2 mL of physiological saline (0.9% NaCl). - Adjust the McFarland value to 0.5 with a nephelometer. - Set a 45 mm black MCE membrane with a pore size of 0.45 μm (HABG047S6, manufactured by Millipore) on a filtration lamp (manufactured by DiaMedix). - Set a funnel. - Prepare a solution by adding 1 mL of bacteria with a McFarland value of 0.5 to 4 mL of physiological water. - Pour this 5 mL solution into the funnel and filter the bacterial solution on the membrane. - Place the membrane on an MH agar medium pre-incubated at 37°C. - Place the E-test with plastic forceps so that no bubbles form, ensuring good diffusion of the antibiotic in the agar. - Incubate at 37°C for 3, 4, or 6 hours with the E-test agar medium facing upwards.

[0135] Marking - After incubation, perform DAPI labeling as follows: · Prepare a 1.4 μg / mL DAPI solution and filter it through 0.22 μm. · Place 700 μL of the 1.4 μg / mL DAPI solution on the glass fiber pad of the petri dish (GE Healthcare Whatman: GF / A ref. 1820 - 047; batch 17076495). · Place the membrane on the glass. · Incubate for 10 minutes at room temperature in the dark. · Wash with 500 mL of phosphate buffer + 0.5 mL of Tween 20 (cfinal = 0.1%) for 15 minutes at room temperature in the dark. The membrane after marking can be stored at 4°C until the next day. - Reading with an optical bench (Diamidex "model 2", magnification: 4, resolution: 12 Mpix) is performed with the following parameters: · UV power: 10 · Exposure time: 262 milliseconds · Gain = 1

[0136] Results For both Escherichia coli (Figure 1) and Staphylococcus aureus (Figure 2), differences in growth between resistant and sensitive strains were observed 3 hours after culturing. A halo was observed around the strip, which intensified with the culture time. The arrows in the figure indicate the minimum growth inhibitory concentration of the antibiotic against the tested strains.

[0137] References [1] International Publication No. 2013 / 130875 of Harvard University, applicant, published on September 6, 2013 [2] International Publication No. 2013 / 107759 of Santorum National de la Recherche Scientifique, applicant, published on July 25, 2013

Claims

1. A method for determining the presence and / or degree of resistance of a microorganism to one or more antibacterial agents, each at one or more concentrations, said method comprising: a. forming a suspension of said microorganism in a liquid medium; b. uniformly attaching the microorganism cells contained in said suspension to at least a part of the upper surface of a membrane; c. placing the lower surface of said membrane on a culture medium; d. placing one or more substrates, each impregnated with one of said antibacterial agents, on the upper surface of said membrane to which the microorganism cells are attached; e. incubating the whole under conditions suitable for promoting the growth of said microorganism; f. contacting the lower surface of said membrane with a fluorescent compound capable of labeling an endogenous component of said microorganism in order to label said microorganism cells; g. optionally, washing the lower surface of said membrane to remove excess fluorescent compound; h. detecting the presence or absence of fluorescence around the substrate on the upper surface of said membrane using a fluorescence reading optical device; i. determining the presence and / or degree of resistance of said microorganism to said antibacterial agent based on the detection of the presence and / or size of a non-fluorescent region around said substrate.

2. Said suspension has a McFarland value of 0.4 to 0.6, and said incubating step is carried out for 3 to 6 hours. The method according to claim 1.

3. Said substrate is one or more discs impregnated with said antibacterial agent at a single concentration and / or one or more elongated strips impregnated with said antibacterial agent having a concentration gradient. The method according to claim 1 or 2.

4. At least one of said substrates is a disc impregnated with one of said antibacterial agents at a single concentration. The method according to any one of claims 1 to 3.

5. At least one of said substrates is a strip impregnated with one of said antibacterial agents having a concentration gradient along the length of the strip. The method according to any one of claims 1 to 3.

6. Determining the minimum inhibitory concentration (MIC) of said antibacterial agent against said microorganism. The method according to any one of claims 1 to 5.

7. Said substrate is made of paper or plastic. The method according to any one of claims 1 to 6.

8. Said optical device has a magnification between 1 and 50 and an optical resolution composed between 6 and 20 Mpx. The method according to any one of claims 1 to 7.

9. The method according to any one of claims 1 to 8, wherein the light source of the optical device includes one or more LEDs.

10. The method according to any one of claims 1 to 9, wherein at least one of the antibacterial agents is an antibiotic.

11. The method according to any one of claims 1 to 10, wherein the microorganism is selected from yeast, fungi, algae, Gram-positive bacteria, Gram-negative bacteria, archaea, and mixtures thereof.

12. The method according to any one of claims 1 to 11, wherein the membrane is made of polytetrafluoroethylene (PTFE), polyester, polycarbonate, nylon, polyvinylidene fluoride (PVDF), cellulose, or a cellulose derivative such as cellulose acetate, cellulose nitrate, regenerated cellulose, nitrocellulose, cellophane, or mixed cellulose ester (MCE).

13. The method according to any one of claims 1 to 12, wherein the fluorescent compound is a compound capable of labeling mitochondria, cell membranes, or nucleic acids.

14. The method according to any one of claims 1 to 13, wherein the fluorescent compound is 4',6-diamidino-2-phenylindole (DAPI).

15. A method for analyzing a sample for at least one microorganism characteristic, the method comprising: detecting the presence of at least one microorganism in the sample; optionally, identifying the microorganism; recovering the microorganism from the sample; performing the method according to any one of claims 1 to 14 to determine the presence and / or degree of resistance of the microorganism to one or more concentrations of one or more antibacterial agents for the recovered microorganism.

16. The sample according to claim 15 is any one of a biological fluid; a plant fluid such as sap, nectar, and exudate from roots; a sample in a culture medium such as a cell culture of higher eukaryotes, yeast, fungi, algae, or in a biological culture reactor; a liquid obtained from one (or more) animal or plant cells; a liquid sample obtained from an animal or plant tissue; a liquid sample obtained from a food matrix; a liquid sample from a chemical reactor; municipal water, river water, pond water, lake water, sea water, or water from an air-cooled tower; a sample from liquid industrial waste, particularly wastewater from intensive animal production or industries in the chemical, pharmaceutical, cosmetic, and nuclear fields; a liquid sample from a pharmaceutical; a liquid sample from a cosmetic; a fragrance; a soil sample; or a mixture thereof.

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