Method for determining presence and / or extent of resistance of microorganisms to antimicrobial agents

Abstract

A method of determining the presence and / or extent of resistance of a microorganism to one or more antimicrobial agents is presented.

Description

Technical Field

[0001] The present invention belongs to the field of microorganisms, and more specifically to the field of qualitatively / quantitatively determining the antimicrobial susceptibility of microorganisms, especially bacteria.

[0002] The present invention proposes a method for determining the presence and / or extent of resistance of a microorganism to one or more antimicrobial agents. The present invention also relates to a more comprehensive method for analyzing the microbial characteristics of a sample, comprising determining the presence and / or extent of resistance of a microorganism to one or more antimicrobial agents by the method of the present invention. Background Art

[0003] In the field of medicine, in the food, biotechnology and pharmaceutical industries, for military and civilian defense and environmental control, the repeated and inappropriate use of antibiotics tends to lead to microbial resistance to these molecules. The result of this inappropriate use is that it is increasingly difficult to fight some microorganisms, especially some bacterial infections.

[0004] In particular, in the therapeutic field, when a microorganism is detected in a patient's biological sample, it is desirable to determine which antibiotics the microorganism is susceptible to. There are now a large number of bacterial species that increasingly show resistance to one or more types of antimicrobial agents, making it even more important to perform antibiotic susceptibility testing in order to better determine which antibiotic is best to administer to a patient.

[0005] For example, in the case of treating a urinary tract infection (UTI), a microbiological analysis of the patient's urine is required.

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

[0007] Symptoms from the lower urinary tract include painful urination, frequent urination, and a sense of needing to urinate even when the bladder is empty. Symptoms of kidney infection include fever and flank pain, often with symptoms of a lower UTI. In some cases, blood may be present in the urine. In the very old and very young, symptoms can be vague or nonspecific. A common cause of infection is the presence of pathogenic Escherichia coli in the urinary tract, but other bacteria, viruses, or fungi can also be the cause.

[0008] UTIs are common infections that are usually treated with antibiotics and can lead to serious complications, such as kidney failure. To avoid these complications and overuse of antibiotics, effective and well-targeted treatments are particularly important.

[0009] Prior art has proposed methods for determining the susceptibility of microorganisms to antibiotics and antimicrobial agents in general. However, these methods are time-consuming, expensive and / or lack sensitivity, and none of them is completely satisfactory.

[0010] Therefore, there is still a need for a method for determining the presence and / or extent of resistance of microorganisms to antimicrobial agents at low cost, sensitively and quickly. The present invention aims to provide such a method. In particular, the object of the present invention is to be able to carry out the method without using any expensive equipment, in particular any expensive optical equipment. Summary of the invention

[0011] The present invention proposes a method for determining the presence and / or degree of resistance to an antimicrobial agent using a universal marker, namely a fluorescent compound and a membrane having an upper surface and a relative lower surface with well-defined different functions. In practice, the microorganisms are deposited on the upper surface and developed thereon, while the lower surface is in continuous contact with the culture medium, the fluorescent compound and the washing solution. Another advantage of the present invention is that no fixation is required to scan the microorganisms marked with the fluorescent compound. This lack of fixation allows the steps of marking with the fluorescent compound, washing and scanning to be repeated as many times as desired.

[0012] Detection methods using fluorescent compounds are well known in the art, but as already mentioned, a fixation step is required and they are usually used for microorganisms in the form of single cells, on which the fluorescent compound is deposited. The present invention applies microorganisms in the form of colonies and / or cell layers on the upper surface of the membrane, and the fluorescent compound is in contact with the microorganism via the lower surface of the membrane. It should be noted that before the present invention, it was not at all obvious whether the effect achieved on single cells was suitable for colonies, and whether the fluorescent compound could diffuse evenly and sufficiently for observation within a short contact time without the need for sophisticated and expensive equipment.

[0013] Furthermore, due to the marking of the microorganisms and the washing via the lower surface of the membrane, there is no risk of eliminating microbial colonies and no fixation is necessary.

[0014] In this application, all ranges must be interpreted as inclusive limitations.

[0015] The object of the present invention is to provide a method for determining the presence and / or extent of resistance of a microorganism to one or more antimicrobial agents, each at one or more concentrations, comprising the consecutive steps of:

[0016] a. forming a suspension of said microorganism in a liquid medium,

[0017] b. uniformly depositing the microbial cells contained in the suspension on at least a portion of the upper surface of the membrane,

[0018] c. laying the lower surface of the membrane on the culture medium,

[0019] d. placing one or more substrates, each impregnated with one of the antimicrobial agents, in contact with the membrane, preferably, the one or more substrates, each impregnated with one of the antimicrobial agents, are placed on the upper surface of the membrane on which the microbial cells have been deposited,

[0020] e. incubating the whole under conditions suitable for promoting the growth of the microorganism,

[0021] f. contacting the lower surface of the membrane with a fluorescent compound to mark the microbial cells, wherein the fluorescent compound is capable of marking endogenous components of the microorganism,

[0022] g. optionally washing the lower surface of the membrane to eliminate excess fluorescent compounds,

[0023] h. detecting the presence or absence of fluorescence around the substrate on the upper surface of the film using a fluorescence reading optical device,

[0024] i. and determining the presence and / or extent of resistance of the microorganism to the antimicrobial agent based on detecting the presence and / or size of the non-fluorescent area around the substrate.

[0025] "Resistance" refers to the ability of a microorganism to resist the action of the antimicrobial agent.

[0026] "Microorganism" refers to microorganisms, especially bacteria, viruses or fungi.

[0027] The microorganisms tested for sensitivity to one or more antimicrobial agents according to the method of the invention are organisms of the eukaryotic type and in particular unicellular organisms or organisms of the prokaryotic type and in particular unicellular organisms.

[0028] The eukaryotic microorganism may be a yeast, such as a yeast of the genus Saccharomyces or a Candida, such as Candida albicans, a fungus, an algae or a mixture thereof.

[0029] The prokaryotic type of microorganism is a bacterium, which may be a Gram-positive type or a Gram-negative type, an archaea or a mixture thereof.

[0030] Thus, the microorganism tested for sensitivity to one or more antimicrobial agents according to the method of the invention is advantageously selected from yeasts, fungi, algae, Gram-positive bacteria, Gram-negative bacteria, archaea and mixtures thereof.

[0031] Among these bacteria, mention is made by way of example and in a non-exhaustive manner of bacteria belonging to the branches of spirochetes and chlamydiae, bacteria belonging to the families or genera of Alcaligenaceae, Pasteurellaceae, Enterobacteriaceae, Enterococcus, Staphylococcus, Pseudomonadaceae, Streptococcus, Micrococci, Legionellae, Mycobacteria, Bacillaceae and Cyanobacteria. More specifically, these bacteria may be selected from 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.

[0032] Preferably, the microorganism is Escherichia coli, Pseudomonas, Candida and / or Staphylococcus.

[0033] From the Archaea, mention may be made, by way of example and in a non-exhaustive manner, of those belonging to the phyla Crenarchaeotes and Euryarchaeotes.

[0034] Of course, the method of the present invention can be applied to mixtures of microorganisms. Therefore, in this specification, the term "microorganism" also covers a mixture of microorganisms, which are then evaluated for their overall sensitivity to antimicrobial agents by the method described in the present invention.

[0035] "Antimicrobial agent" refers to a natural or synthetic substance that kills or inhibits the growth of microorganisms such as bacteria, fungi, and algae.

[0036] Among the antimicrobial agents, mention may be made, by way of example and in a non-exhaustive manner, of antibacterial agents, antifungal agents, antiviral agents, antiparasitic agents, broad-spectrum therapeutic agents, non-pharmaceutical antimicrobial agents (e.g. essential oils and antimicrobial insecticides), ozone, antimicrobial agents, halogens, alcohols, phenols and phenolic compounds.

[0037] Preferably, the antimicrobial agent according to the present invention is antibacterial.

[0038] In a preferred embodiment of the invention, at least one, and preferably all, antimicrobial agents are antibiotics.

[0039] In step (a) according to the present invention, a suspension of microorganisms in a liquid medium is formed.

[0040] Thus, any suitable method may be implemented according to the present invention.For example, the microorganism may be transferred from its original culture medium to a liquid culture medium.

[0041] "Original culture medium" refers to any liquid or solid sample that may contain microorganisms after contamination.

[0042] Preferably, the original culture medium according to the present invention is a liquid sample.

[0043] Typically, the fluid sample can be a biological fluid; a plant fluid, such as plant sap, nectar and root exudate; a sample in a culture medium or in a bioreactor, such as a cell culture of higher eukaryotes, yeast, fungi or algae; 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; water from a city, river, pond, lake, ocean or air cooling tower; a sample from a liquid industrial effluent; in particular wastewater from intensive animal production or industry in the chemical, pharmaceutical, cosmetic or 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.

[0044] Within the scope of the present invention, the sample may be obtained by any type of sample collection, such as by contact, scraping, drilling, draining, washing, flushing, aspirating, pumping, etc.

[0045] When the liquid sample is a biological fluid, the latter is advantageously selected from blood, such as whole blood or anticoagulated whole blood, serum, plasma, lymph, saliva, sputum, tears, sweat, sperm, urine, feces, milk, cerebrospinal fluid, interstitial fluid, the fluid separated from the bone marrow, mucus or fluid separated from the respiratory tract, intestinal tract or urogenital tract, cell extract, tissue extract and organ extract. Therefore, the biological fluid can be any fluid naturally secreted or excreted from the human body or animal body, or by any technology known to those skilled in the art, such as extracting, sampling or washing any fluid recovered from the human body or animal body. The step of reclaiming and separating these different fluids from the human body or animal body is carried out before implementing the method according to the present invention. Preferably, the liquid sample is urine.

[0046] Likewise, if one of the intended samples does not allow the implementation of the method according to the invention, for example due to its nature, in particular solid, its concentration or the fact that it contains elements such as solid residues, waste, suspensions or interfering molecules, then said sampling as defined below also comprises a previous step of preparing said sample by techniques known to those skilled in the art, such as filtration, precipitation, dilution, distillation, mixing, concentration, lysis, etc., optionally dissolving said sample.

[0047] For example, when the liquid sample is urine, the latter can be filtered to remove interfering substances, such as crystals, hyaluronic acid structures, cell aggregates and / or large eukaryotic cells. For this purpose, a sterile sieving device with a porosity of 5 μm or 10 μm can be used, such as However, such filtering is optional.

[0048] Preferably, the suspension of microorganisms in liquid culture medium formed according to the invention is prepared from isolated colonies.

[0049] Preferably, the suspension of microorganisms in liquid culture medium formed according to the present invention has a McFarland value between 0.4 and 0.6.

[0050] "McFarland value" refers to the approximate number of bacteria in a standardized liquid suspension by comparing the turbidity of the test suspension to the turbidity of a McFarland standard. The turbidity of a microbial suspension is proportional to the number of microbial cells in the suspension that is within the relevant range for standardized microbial tests.

[0051] Preferably, the suspension according to step (a) of the present invention has a McFarland value of about 0.5.

[0052] In a specific embodiment of the present invention, the liquid culture medium is a nutrient medium for the microorganism, such as liquid lysozyme broth (LB) medium or Mueller Hinton (MH) broth or physiological saline (NaCl 0.9%), and the culture medium is inoculated with the microorganism and incubated under conditions that promote the growth of the microorganism until a sufficient number of cells is reached to obtain a desired McFarland value.

[0053] Any other means of forming a microbial suspension having a McFarland value between 0.4 and 0.6 falls within the scope of the present invention, such as diluting an already available overconcentrated cell suspension resulting from, for example, a previous step of detecting and / or identifying microorganisms in the original sample.

[0054] "Membrane" refers to a thin wall of a porous substance that is inserted between two media and allows the removal or concentration of certain components.

[0055] Obviously, the membrane implemented in the present invention is capable of retaining the microorganisms contained in the suspension and / or is impermeable to the microorganisms. Advantageously, the 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, the porosity is less than 0.5 μm, in particular 0.45 μm or less.

[0056] Any film that is usually used for filtering liquid samples and retaining the described microorganism that exists therein can be implemented for the present invention. Suitable film for the present invention includes but is not limited to the film made of polytetrafluoroethylene (PTFE), polyester, polycarbonate, nylon, polyvinylidene fluoride (PVDF), cellulose and cellulose derivatives such as cellulose acetate, nitrocellulose, regenerated cellulose, nitrocellulose, celluloid or mixed cellulose ester (MCE). Such film can be obtained from Waterman Company, Merck Millipore Corporation and Sigma-Aldrich Corporation especially.

[0057] Advantageously, the membrane implemented in the present invention is an MCE membrane, in particular a black MCE membrane, more particularly a black MCE membrane No. 1 sold by Merck Millipore.

[0058] It should be noted that the membrane implemented in the present invention does not have to be pretreated before step (b). The pretreatment may be a coating, in particular a coating with microorganism binding molecules as defined in [1]. Preferably, the membrane implemented in the present invention is not pretreated before step (b).

[0059] Step (b) of the method according to the present invention comprises uniformly depositing the microbial cells contained in the suspension on at least a portion of the upper surface of the membrane, in which step the membrane may or may not have been laid on the culture medium via its lower surface.

[0060] This deposition step can be carried out, for example, by filtering at least part of the suspension on the membrane to collect the microbial cells thereon. In such an embodiment, it is preferred that the membrane is not yet located on the substratum. For example, it can be carried out by depositing the suspension of a small volume on the membrane and using beads or any other conventional means that the deposited cells are evenly spread on the membrane. In such an embodiment, it is preferred that the membrane is already located on the substratum.

[0061] "Media" refers to a nutrient-containing substance in which microorganisms can grow.

[0062] In a first embodiment, the culture medium implemented in step (c) of the method according to the present invention is a liquid culture medium.

[0063] The liquid culture medium implemented in step (c) of the method according to the invention may be any liquid culture medium suitable for cultivating microorganisms known to the skilled person. The latter has a carbon source, such as glucose or glycerol; a nitrogen source, such as ammonium or nitrates or amino acids; and salts and / or trace elements and vitamins for the growth of microorganisms. In particular, the liquid culture medium may not be selective for a specific type of microorganism. Alternatively, it may be selective for a specific type of microorganism.

[0064] As examples of liquid culture media suitable for step (c) of the method of the present invention, Nutrient Broth No. 1 (NB1), Nutrient Broth No. 2 (NB2), Mueller Hinton Broth, Lysozyme Broth (LB) (also known as Luria Bertani Broth), Tryptic Soy Bean (TSB) broth and Brain Heart Infusion (BHI) broth can be cited. Such culture media can be obtained from Thermo Fisher Scientific, Bio-Rad and Sigma-Aldrich.

[0065] These various liquid media may be supplemented with one or more elements selected from the group consisting of: antioxidants, such as sodium pyruvate, glutamate, calcium chloride and magnesium chloride; multivitamins, such as (PVX); an iron source such as ferric citrate or protein supplements, such as bovine serum albumin (BSA); and anti-caking agents, such as citrate.

[0066] Advantageously, the liquid culture medium is sterile or has been sterilized before contacting the lower surface of the membrane in step (c) of the method according to the invention. The culture medium can be sterilized by various techniques known in the art, such as, but not limited to, autoclaving and / or preparing the culture medium using aseptic techniques.

[0067] In a second embodiment, the culture medium implemented in step (c) of the method according to the present invention is a solid culture medium.

[0068] In this second embodiment, the solid culture medium may be a diffusion intermediate impregnated with a liquid culture medium such as previously defined. A diffusion intermediate is a solid support which must be porous, allowing good impregnation with a liquid culture medium such as previously defined. Advantageously, the diffusion intermediate is a porous solid support, such as a pad in a material selected from: paper, in particular of a cellulose nature; cotton paper; agarose; gelatin; cellulose; methylcellulose; carboxymethylcellulose; nitrocellulose; cellulose acetate; alginates; polyolefins; porous membranes, in particular ion exchange porous membranes; sephadex-type resins packaged into membranes or perfluoropolymers, such as PVDF membranes; felt fabrics; glass fiber fabrics; organic polymers, such as films in polyethylene, polypropylene or mixtures thereof; nylon fabrics; polyacrylamide gels; agarose gels and mixtures thereof.

[0069] As a variation of this second embodiment, the solid culture medium may be a nutrient gel. The latter may be any nutrient gel known to those skilled in the art that is suitable for culturing microorganisms. For liquid culture medium, the nutrient gel has a carbon source, such as glucose or glycerol; a nitrogen source, such as ammonium or nitrates or amino acids; and salts and / or trace elements and vitamins for microbial growth. In particular, the solid culture medium may not be selective for a specific type of microorganism. Alternatively, it may be selective for a specific type of microorganism.

[0070] As examples of solid culture media suitable for step (c) of the method according to the invention, nutrient agar, tryptone soy agar (TSA), Mueller Hinton agar, Legionella glycine vancomycin polymyxin cyclohexamide (GVPC) agar, buffered charcoal yeast extract (BCYE) agar, buffered cephalexin-polymyxin-anisomycin α-acetylgluconate (BMPAα) agar, Reasoner's 2A (R2A) agar, Sabouraud dextrose agar (SDA), Columbia horse blood agar, blood agar such as sheep blood agar, chocolate agar supplemented with Vitox, Haemophilus test medium (HTM) agar and charcoal agar may be cited. Such culture media may be obtained in particular from Thermo Fisher Scientific, Oxoid and VWR.

[0071] Advantageously, when the culture medium in step (c) of the present invention is solid, it is blood agar.

[0072] Furthermore, when a solid culture medium is used, some liquid culture medium, such as the liquid culture medium defined above, may be added between the solid culture medium and the membrane, the upper surface of which is the microorganism. However, such addition is optional.

[0073] Step (d) of the method according to the present invention comprises placing one or more substrates on the portion of the upper surface of the membrane on which the microbial cells have been deposited, wherein each of the one or more substrates is impregnated with one of the antimicrobial agents.

[0074] "Substrate" refers to any carrier capable of at least partially absorbing an antimicrobial agent and allowing it to diffuse into its environment.

[0075] In one embodiment, step d is performed before step b or before step c. In this case, the antimicrobial agent diffuses from the substrate through the membrane to the surface of the membrane. "Before step b" preferably means that step d is performed between steps a and b. In this case, before "placing one or more substrates impregnated with one of the antimicrobial agents in contact with the membrane", step d also includes the sub-step of "providing a membrane". "Before step c" preferably means that step d is performed between steps b and c.

[0076] In another embodiment, step d is performed after step c. In this case, the antimicrobial agent diffuses from the substrate onto the upper surface of the membrane to form an antimicrobial gradient thereon. This other embodiment is preferred.

[0077] During the subsequent step (e) of the method according to the present invention, the inhibition of the growth of the microorganism on the film around the substrate indicates the susceptibility of the microorganism to the antimicrobial agent. The larger the growth inhibition zone, the lower the degree of resistance of the microorganism to the antimicrobial agent. The absence of any growth inhibition zone on the film indicates the resistance of the microorganism to the tested antimicrobial agent.

[0078] According to the method of the present invention, a single substrate can be used to assess the susceptibility of the microorganism to a single antimicrobial agent that impregnates the substrate. It can additionally use a plurality of substrates, each of which is impregnated with different antimicrobials, to assess the susceptibility of the microorganism to the multiple antimicrobials. In such an embodiment of the present invention, as suggested by the laboratory test guide, the substrates are preferably separated from each other and placed on the film to interfere with each other.

[0079] The substrate according to the implementation of the present invention is an antibiotic-impregnated disk and / or an elongated strip impregnated with a gradient antibiotic. Preferably, the substrate according to the implementation of the present invention is an antibiotic-impregnated disk or an elongated strip impregnated with a gradient antibiotic ( In particular, in these embodiments, the method according to the invention allows determining the presence and extent of resistance of a microorganism to one or more antibiotics.

[0080] In a particular embodiment of the method according to the invention, at least one substrate, preferably all substrates, are discs which are each impregnated with a single concentration of the antimicrobial agent.

[0081] In an embodiment of the method according to the invention, at least one substrate, preferably all substrates, is a strip impregnated with a concentration gradient of the antimicrobial agent along its length.

[0082] Advantageously, the substrates according to the implementation of the invention are made of paper or plastic. Preferably, they are made of plastic.

[0083] Preferably, the substrate according to the practice of the invention is a strip impregnated with a gradient antibiotic, for example under the trade name The strips are made of thin, inert, non-porous plastic slides with a dilution range following a predetermined antibiotic gradient on one side and a scale for MIC readings in μg / mL on the other side.

[0084] The step (e) of the method according to the present invention is included in hatching all under conditions suitable for promoting the growth of the microorganism, that is, the microorganism is maintained in an environment that allows it to increase in number by cell division, so as to form a microcolony, a colony or even a cell layer. This environment not only includes the culture medium contacted with the lower surface of the film, but also includes other conditions, such as the temperature and atmosphere that allow cell growth. Typically, the temperature implemented during step (e) is included between 25 ° C and 40 ° C. The special example of the temperature implemented in step (e) is 30 ° C, 32 ° C, 37 ° C and 39 ° C. Typically, with regard to atmospheric conditions, the step (e) can be carried out under air, under air + 5% CO2 or under a gas mixture consisting of 5% O2, 5% CO2 and 90% N2.

[0085] Typically, step (e) must last no longer than 8 hours, in particular no longer than 7 hours, in particular no longer than 6 hours, more in particular no longer than 5 hours, depending on the type of microorganisms present in the liquid sample. In particular, step (e) must last at least 2 hours, in particular at least 2.5 hours, in particular at least 3 hours. In practice, for example, from the results obtained by the inventors, in particular under the conditions disclosed in the experimental part below, it is possible to detect colonies of Escherichia coli (E. coli) or Staphylococcus aureus (S. aureus) after 4 hours of incubation with the culture medium.

[0086] In a particularly advantageous embodiment of the present invention, said suspension formed in step (a) has a McFarland value between 0.4 and 0.6, and said incubation step (e) is carried out for a duration of 3 to 6 hours.

[0087] The step (f) of the method according to the present invention is a labeling step for labeling the microbial cells that have grown onto the upper surface of the membrane obtained after steps (b) to (e) with a fluorescent compound.

[0088] In general, the term "fluorescent compound" refers to a compound that is capable of emitting light when excited by another light of an appropriate wavelength. When applied to a compound, the term "fluorescence" can be used to refer to its property of absorbing energy (e.g., UV, visible light, or IR radiation) and re-emitting at least a portion of that energy as light over time.

[0089] The present invention implements fluorescent compounds capable of recognizing and binding to endogenous components of the microorganism. By definition, endogenous components are components originating from within the microorganism.

[0090] Typically, the fluorescent compound implemented in step (f) of the method according to the present invention can recognize and bind to, thereby marking mitochondria, cell membranes or nucleic acids. Alternatively, the fluorescent compound implemented in step (f) of the method according to the present invention can also mark cytoplasm.

[0091] In a first embodiment, the fluorescent compound implemented in step (f) of the method according to the invention is capable of recognizing and binding to mitochondria, thereby marking mitochondria. Advantageously, the fluorescent compound is selected from Rhodamine 123, DiOC 6 (3), DiOC 7 (3), JC-1, MitoTracker ORANGE and Red CMXRos.

[0092] In a second embodiment, the fluorescent compound implemented in step (f) of the method according to the invention is capable of recognizing and binding to cell membranes, thereby marking the cell membranes. Advantageously, the fluorescent compound is selected from FM1-43 and FM4-64.

[0093] In a third embodiment, the fluorescent compound implemented in step (f) of the method according to the invention is capable of recognizing and binding to nucleic acids, thereby labeling nucleic acids. "Nucleic acid" is understood to mean single-stranded or double-stranded deoxyribonucleic acid (DNA), ribonucleic acid (RNA), such as messenger RNA or ribosomal RNA.

[0094] The fluorescent compound specific for nucleic acids is in particular selected from fluorescent intercalators, dyes binding the bases A:T or the bases G:C and permeating or non-permeating anthocyanins. More particularly, the fluorescent compound is selected from ethidium bromide, thiazole orange, thiazole blue and its derivatives, thioflavin S, thioflavin T, thioflavin TCN, diethylquinolylthio-cyanine iodide (DEQTC), TOTO1, TOPRO1, TOTO3, TOPRO3, YOYO1, Hoechst 33258, Hoechst33342, Hoechst34580, 4',6-diamidino-2-phenylindole (DAPI), pyrene Y, acridine orange, auramine O, calcein, olamin-O, oxazine 750, star blue and the SYTO series, in particular including SYTO 11, SYTO 12, SYTO 13, SYTO 15, SYTO 16, SYTO 18, SYTO 62, SYTO 80 or SYTO 81.

[0095] The fluorescent compound can be selected from 4',6-diamidino-2-phenylindole (DAPI), rhodamine 123, 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, Hoechst34580, acridine orange, calcein, olamin-O, oxazine 750, star blue and the SYTO series, in particular including SYTO 11, SYTO 12, SYTO 13, SYTO 15, SYTO 16, SYTO 18, SYTO 62, SYTO 80 or SYTO 81.

[0096] In a particular embodiment, the fluorescent compound implemented in step (f) of the method according to the present invention is 4',6-diamidino-2-phenylindole (DAPI).

[0097] In a fourth embodiment, the fluorescent compound implemented in step (f) of the method according to the present invention is capable of labeling the cytoplasm.

[0098] In a particular embodiment of this fourth embodiment, the fluorescent compound implemented in step (f) can be released from a molecular probe, which collapses in response to a stimulus, such as the presence of an enzyme, to release the fluorescent compound. For example, to label the cytoplasm, the molecular probe can be 5(6)-CFDA (5-(and-6)-carboxyfluorescein diacetate) of alanine aminopeptidase commercialized by Molsid or SmartID TM Green probe.

[0099] During this step (f), the fluorescent compound is brought into contact with the lower surface of the membrane, thereby rendering it permeable.

[0100] In a particular embodiment, the lower surface of the membrane may be contacted with a solution or dispersion of the fluorescent compound. The fluorescent compound is preferably present in the solution or dispersion in an excess amount relative to the microorganisms present on the upper surface of the membrane.

[0101] In another particular embodiment, the lower surface of the membrane can be contacted with a solution or dispersion of the fluorescent compound through a diffusion intermediate impregnated with the solution or dispersion, the fluorescent compound being as previously defined. The diffusion intermediate is as previously defined for the solid culture medium, with the proviso that in step (e), this diffusion intermediate is a solid support that must be porous in order to allow good impregnation with the solution or dispersion of the fluorescent compound. A particular example of such a diffusion intermediate that can be used in step (f) is a glass fiber fabric impregnated with a solution or dispersion of a fluorescent compound, such as a DAPI solution, more particularly including a 1.4 μg / ml solution.

[0102] Typically, the duration of step (f) of the method according to the invention is equal to or lower than 30 minutes, advantageously equal to or lower than 25 minutes, in particular equal to or lower than 20 minutes, in particular equal to or lower than 15 minutes, more in particular 10 minutes (i.e. 10 minutes ± 2 minutes).

[0103] Said step (f) of the process according to the invention may be carried out at a temperature between 10°C and 40°C, advantageously between 15°C and 30°C, more particularly at room temperature (ie 23°C±5°C).

[0104] In particular, said step (f) of the method according to the present invention can be performed in the dark.

[0105] The step (g) of the method according to the invention is an optional washing step which allows the removal of any fluorescent compound present on the lower surface of the membrane. In fact, the presence of the fluorescent compound adsorbed on the lower surface of the membrane may generate background noise during the step (h).

[0106] The lower surface of the membrane is subjected to at least one rinse in order to remove all traces of the fluorescent compound and / or a solution or dispersion containing it. The rinse is generally performed with a rinse solution, in particular an aqueous rinse solution that does not affect the microbial cells on the upper surface of the membrane. The solution may also help to eliminate nonspecific interactions. It may contain one or more of the following components: a buffer, such as Tris, phosphate, acetate or borate buffer; a salt, such as KCl, NaCl, (NH4)2SO4, MgCl2 or CaCl2; and a detergent or surfactant, such as Sodium dodecyl sulfate (or SDS) or bovine serum albumin (BSA). For each rinse, the rinse can be repeated two, three, five, 10 or even 50 times using the same or different rinse solutions. Typically, a phosphate buffered saline (or PBS) and 0.1% And the washing was repeated three times with a solution having a pH between 7 and 7.5.

[0107] Advantageously, the washing step (g) can be carried out by causing the washing solution to flow continuously at the level of the lower surface of the membrane. This can be carried out using washing devices for membranes known in the art, for example Washing modules for sale.

[0108] Typically, the duration of step (g) of the process according to the invention is equal to or lower than 40 minutes, advantageously equal to or lower than 30 minutes, in particular equal to or lower than 20 minutes, in particular 15 minutes (ie 15 minutes ± 3 minutes).

[0109] Said step (g) of the process according to the invention may be carried out at a temperature between 10°C and 40°C, advantageously between 15°C and 30°C, in particular at room temperature (ie 23°C±5°C).

[0110] In particular, said step (g) of the method according to the present invention is carried out in the dark.

[0111] The step (h) of the method according to the invention consists in placing the film obtained after step (g) in an optical device capable of reading fluorescence, such as under a microscope, in which the film can be tested by an excitation source suitable for the fluorescent compound being implemented.

[0112] In an embodiment of the method implemented according to the invention, images of the film are captured at one or more intervals with at least one image capture device.

[0113] Typically, the excitation source results in excitation of the fluorescent compound labeling the microorganism, followed by measurement of fluorescent emission from the compound.

[0114] The membrane may be manipulated manually to position the substrate of step (d) for testing, either by moving the membrane itself or by moving the optical device on which the membrane is placed. Alternatively, the optical device is automatically controlled so that the membrane on the stage can be scanned.

[0115] Light sources that can emit in ultraviolet, visible and / or near infrared spectra well known to those skilled in the art can be used for the present invention.For example, the light source can be a continuous lamp, such as a deuterium or xenon arc lamp for producing ultraviolet light and a tungsten halogen lamp for producing visible / near infrared excitation.Or, the light source can be one or more LEDs.These light sources provide a wide emission range, and the optical filter of, for example, an optical interference filter, a prism and / or a grating can be used to reduce the spectral bandwidth of a specific excitation wavelength.

[0116] Therefore, in a particular embodiment of the invention, the light source in the optical device comprises one or more LEDs.

[0117] The skilled person knows which excitation source to use depending on the fluorescent compound implemented in the method according to the invention. Advantageously, the excitation source implemented at step (h) of the method according to the invention is an optical microscope equipped with filters such as UV filters or Cy5 filters.

[0118] The measurement data obtained when performing step (h) can be analyzed by an algorithm, in particular compared with comparative measurement data.

[0119] Typically, the duration of step (h) of the process according to the invention is equal to or lower than 20 minutes, advantageously equal to or lower than 15 minutes, in particular equal to or lower than 10 minutes, in particular 7 minutes (ie 7 minutes ± 2 minutes).

[0120] Taking into account the duration of the different steps of the method according to the invention, the latter steps (b) to (i) are carried out in less than 10 hours, in particular in less than 9 hours, preferably in less than 8 hours, more preferably in less than 7 hours, even more preferably in less than 6 hours, and even more preferably in less than 5 hours.

[0121] The optical device involved in step (h) of the present invention is a simple and inexpensive optical device.

[0122] In one embodiment, the optical device of step (h) has a magnification between 1 and 50 and an optical resolution between 6 and 20 Mpix.

[0123] Preferably, the magnification of the optical device is between 1 and 8, more preferably between 1 and 4, such as between 2 and 4.

[0124] Preferably, the optical resolution of the optical device is between 9 and 15, more preferably between 11 and 13 Mpix.

[0125] The presence and / or extent of resistance of the microorganism to the antimicrobial agent is determined in step (i) of the method of the present invention based on the absence or presence and optionally the size of the non-fluorescent zone detected around the substrate. Determining the extent of resistance of the microorganism to the antimicrobial agent based on the size of the growth inhibition zone is within the skill of a person skilled in the art.

[0126] In particular, for all types of microorganisms, the present invention allows determining the presence and / or extent of resistance of said microorganisms to said antimicrobial agents by shortening the duration of said method by a factor of 4 to 5.

[0127] More particularly, when using elongated strips impregnated with a gradient of antibiotics, the method according to the invention allows determining the presence and degree of resistance of microorganisms to said antibiotics by simply reading the pictures taken by said optical device (see Figure 3 ).

[0128] In one embodiment, the method according to the present invention comprises determining the minimum inhibitory concentration (MIC) of said antimicrobial agent for said microorganism.

[0129] "Minimum inhibitory concentration (MIC)" refers to the lowest concentration of a chemical, usually a drug, that prevents visible growth of the target microorganism.

[0130] The MIC depends on the microorganism and the antimicrobial agent itself. It is usually expressed in micrograms per milliliter (μg / mL) or milligrams per liter (mg / L).

[0131] Advantageously, in the method of the present invention, a strip containing a predetermined and continuous gradient of antibiotic concentrations is used (e.g. or equivalent) to assess MIC.

[0132] Preferably, the strip is graded and indicates the concentration of antibiotic present at all heights of the strip, which aids in the interpretation of the results.

[0133] In order to obtain the inhibition ellipse for determining MIC, according to the present invention, the strip is simply placed on the film of the inoculation and incubated. After the incubation period, the MIC value (in μg / mL) can be read directly on the scale at the intersection between the inhibition ellipse and the strip. This MIC value indicates that the technician will be able to select the antimicrobial agent and its amount for optimal treatment later. In fact, the MIC value read on the strip is compared with the critical concentration defined by the European Antimicrobial Susceptibility Committee Test (EUCAST) for the pathogen / antibiotic pair. If the MIC value read on the strip is lower than the minimum critical concentration, the pathogen is sensitive to the antibiotic at a standard dose; if the MIC value read on the strip is higher than the maximum critical concentration, the pathogen is referred to as resistant to the antibiotic; finally, if the MIC value read on the strip is between the minimum critical concentration and the maximum critical concentration, the pathogen is referred to as moderate or sensitive to the antibiotic at a high dose.

[0134] Another aspect of the present invention relates to a method for analyzing a sample for microbiological properties, the method comprising:

[0135] - detecting the presence of at least one microorganism in said sample,

[0136] - optionally, identifying said microorganism,

[0137] - recovering said microorganism from said sample,

[0138] - and subjecting said recovered microorganisms to a method according to the invention, said method determining the presence and / or extent of resistance of the microorganisms to one or more antimicrobial agents, each at one or more concentrations.

[0139] Said step of detecting the presence of at least one microorganism in said sample and optionally identifying said microorganism may be performed by any method known in the art.

[0140] The sample may be any biological fluid; plant fluids, such as juice, nectar and root exudate; samples in culture medium or in bioreactors, such as cell cultures of higher eukaryotes, yeast, fungi or algae; liquids obtained from one (or more) animal or plant cells; liquid samples obtained from animal or plant tissues; liquid samples obtained from food matrices; liquid samples from chemical reactors; water from cities, rivers, ponds, lakes, oceans or air cooling towers; samples from liquid industrial effluents; in particular wastewater from intensive animal production or industry in the chemical, pharmaceutical, cosmetic or nuclear fields; liquid samples from pharmaceutical products; liquid samples from cosmetic products; spices; soil samples or mixtures thereof. It may respond to any of the above characteristics of the original culture medium containing the microorganism to be tested.

[0141] Other characteristics and advantages of the invention will become apparent to a person skilled in the art upon reading the following examples, which are given by way of illustration and not limitation with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0142] Figure 1 Shown are membrane images of E. coli DSM 1103 strains sensitive to gentamicin or E. coli ATCC BAA 2452 strains resistant to gentamicin taken with a simple optical device at 3 hours (A and E), 4 hours (B and F) or 6 hours (C and G) after starting to cultivate each bacterium in the presence of a strip impregnated with antibiotics (white strips on the image). Images D and H correspond to 24-hour incubation of E. coli DSM 1103 strains and E. coli ATCC BAA 2452 strains directly on MH agar plates with gentamicin impregnated strips. White arrows indicate the intersection between the inhibition ellipse and the strip, which determines the MIC of the strain to gentamicin.

[0143] Figure 2 Shown are membrane images of the oxacillin-sensitive S. aureus DSM 2568 strain or the oxacillin-resistant S. aureus DSM 28766 strain taken with a simple optical device at 3 hours (A and E), 4 hours (B and F) or 6 hours (C and G) after starting the culture of each bacterium in the presence of a strip impregnated with antibiotics (white strips on the images). Images D and H correspond to 24 hours of incubation of the S. aureus DSM 2568 strain and the S. aureus DSM 28766 strain, respectively, in the presence of a strip impregnated with oxacillin directly on MH agar plates. The white arrows indicate the intersection between the inhibition ellipse and the strip, which determines the MIC of the strain to oxacillin.

[0144] Figure 3 Shown is an image of a membrane of the oxacillin-sensitive Staphylococcus aureus (S. aureus) DSM 2568 strain taken with a simple optical setup 3 hours after initiation of incubation in the presence of a strip impregnated with oxacillin. DETAILED DESCRIPTION

[0145] Use of the method according to the invention for evaluating MIC (minimum inhibitory concentration)

[0146] A. Materials and Methods

[0147] sample

[0148] The following bacterial strains were used in this study:

[0149] - Escherichia coli (Gentamycin sensitive) - Ref. Dsm1103

[0150] - Escherichia coli (resistant to gentamycin) - Reference number BAA-2452 TM

[0151] - Staphylococcus aureus (Oxacillin sensitive) - DSMZ 2568

[0152] - Staphylococcus aureus (Oxacillin resistant) - DSMZ 28766

[0153] Description of the resources and equipment used

[0154] equipment:

[0155] The equipment used includes a filter bench, a rinse bench, and an optical bench.

[0156] The filter station is operated with a vacuum pump and can filter 6 samples simultaneously.

[0157] The rinsing station operates continuously with a pump and is able to wash 6 samples simultaneously. The washing is carried out continuously, the washing solution flows between the 6 samples and is recirculated.

[0158] The optical bench was equipped with LEDs and filters enabling reading of either blue fluorescence ("DAPI" channel) or red fluorescence ("FM4-64" channel).

[0159] Impregnation substrate:

[0160] The substrate used was BioMérieux The strips were impregnated with gentamicin (for E. coli experiments) or oxacillin (for Staphylococcus aureus experiments). The references of the e-test used are as follows:

[0161] -ETEST Gentamycin: Ref. 412368

[0162] -ETEST Oxacillin (=Methicillin): Ref. 412432

[0163] Experimental protocol

[0164] J-1: Preparation of bacterial samples

[0165] - Inoculate TSA plates with different bacterial strains using the streak technique (one bacterium - one plate),

[0166] - Incubate at 37°C for 24 hours.

[0167] J0: AST: filter, incubate

[0168] Protocol without filtration step

[0169] - Recover several colonies of the same size and morphology in 2 mL of saline (0.9% NaCl),

[0170] - Use a turbidity meter to adjust the McFarland value to 0.5,

[0171] - Place a 45 mm black MCE membrane (HABG047S6, Millipore) with a porosity of 0.45 μm on the MH agar pre-incubated at 37°C, avoiding the formation of bubbles,

[0172] - Apply 1 mL of a suspension of the corresponding strain with a McFarland value of 0.5 on the MCE membrane,

[0173] - Add 10 sterile glass beads of 2 mm diameter, shake the box up and down 10 times, then shake from left to right 10 times, and finally rotate clockwise and counterclockwise 10 times,

[0174] - Place the E-test with plastic tweezers, avoiding the formation of air bubbles to allow good diffusion of the antibiotic in the agar,

[0175] - Incubate at 37°C for 3, 4 or 6 hours with the E-test agar facing up.

[0176] Alternative protocol with filtration step (not performed in this case)

[0177] - Recover several colonies of the same size and morphology in 2 mL of saline (0.9% NaCl),

[0178] - Use a turbidity meter to adjust the McFarland value to 0.5,

[0179] - a 45 mm black MCE membrane (HABG047S6, Millipore) with a porosity of 0.45 μm was deposited on a filtration ramp (Diamidex),

[0180] - Place the hopper,

[0181] - Prepare 4 mL of physiological aqueous solution, in which there is 1 mL of bacteria with a McFarland value of 0.5,

[0182] - Pour 5mL of solution into the funnel and filter the bacterial solution onto the membrane,

[0183] - Place the membrane on MH agar pre-incubated at 37°C,

[0184] - Place the E-test with plastic tweezers, avoiding the formation of air bubbles to allow good diffusion of the antibiotic in the agar,

[0185] - Incubate at 37°C for 3, 4 or 6 hours with the E-test agar facing up.

[0186] mark

[0187] -After incubation, perform DAPI labeling as follows:

[0188] o Prepare a 1.4 μg / mL DAPI solution, filter at 0.22 μm,

[0189] o Place 700 μL of 1.4 μg / mL DAPI solution on a glass fiber mat in a culture dish (GE Healthcare Whatman: GF / A Ref. 1820-047;

[0190] Batch No. 17076495),

[0191] o Place the film on the glass,

[0192] oIncubate at room temperature in the dark for 10 min.

[0193] o At room temperature in the dark with 500 mL of phosphate buffered saline

[0194] + 0.5 mL Tween 20 (cfinal = 0.1%) and wash for 15 min in a washing station (Diamidex). After labeling, the membrane can be stored at 4°C until the next day

[0195] - On an optical bench (Diamidex "Model 2", magnification: 4, resolution:

[0196] 12Mpix) using the following parameters:

[0197] UV power: 10%

[0198] Exposure time: 262ms

[0199] Gain = 1

[0200] result

[0201] For E. coli ( Figure 1 ) and Staphylococcus aureus (S. aureus) ( Figure 2 ), observed growth differences between resistant strains and sensitive strains cultured for 3 hours. A halo was observed around the band and increased with the time of culture. The arrows in the figure represent the minimum inhibitory concentration of the antibiotics for the tested bacterial strains.

[0202] References

[0203] [1] International application WO 2013 / 130875 A1, filed on behalf of Harvard College, published on September 6, 2013.

[0204] [2] International application WO 2013 / 107759 A1, filed on behalf of the Centre National de la Recherche Scientifique, published on July 25, 2013.

Claims

1. A method for determining the presence and / or extent of resistance of a microorganism to one or more antimicrobial agents, wherein: The one or more antimicrobial agents are each at one or more concentrations, the method comprising: a. forming a suspension of said microorganism in a liquid medium, b. uniformly depositing the microbial cells contained in the suspension on at least a portion of the upper surface of the membrane, c. laying the lower surface of the membrane on the culture medium, d. placing one or more substrates on the upper surface of the membrane on which the microbial cells have been deposited, wherein each of the one or more substrates is impregnated with one of the antimicrobial agents, e. incubating the whole under conditions suitable for promoting the growth of the microorganism, f. contacting the lower surface of the membrane with a fluorescent compound to mark the microbial cells, wherein the fluorescent compound is capable of marking endogenous components of the microorganism, g. optionally washing the lower surface of the membrane to eliminate excess fluorescent compounds, h. using a fluorescence reading optical device to detect the presence or absence of fluorescence around the substrate on the upper surface of the film, and, i. determining the presence and / or extent of resistance of the microorganism to the antimicrobial agent based on the presence and / or size of the detected non-fluorescent area around the substrate.

2. The method according to claim 1, wherein: The suspension had a McFarland value of 0.4 to 0.6 and the incubation step lasted for 3 to 6 hours.

3. The method according to claim 1 or 2, wherein: The substrate is one or more disks impregnated with a single concentration of antimicrobial agent and / or one or more strips impregnated with gradient concentrations of antimicrobial agent.

4. The method according to any one of claims 1 to 3, wherein: At least one of the substrates is a disc impregnated with a single concentration of one of the antimicrobial agents.

5. The method according to any one of claims 1 to 3, wherein: At least one of the substrates is a strip impregnated with a gradient concentration of one of the antimicrobial agents along its length.

6. A method according to any one of the preceding claims, wherein: The minimum inhibitory concentration (MIC) of the antimicrobial agent against the microorganism is determined.

7. A method according to any one of the preceding claims, wherein: The substrate is made of paper or plastic.

8. A method according to any one of the preceding claims, wherein: The optical device has a magnification between 1 and 50 and an optical resolution between 6 and 20 Mpix.

9. A method according to any one of the preceding claims, wherein: The light source in the optical device includes one or more LEDs.

10. A method according to any one of the preceding claims, wherein: At least one of the antimicrobial agents is an antibiotic.

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

12. A method according to any one of the preceding claims, wherein: The membranes are made of polytetrafluoroethylene (PTFE), polyester, polycarbonate, nylon, polyvinylidene fluoride (PVDF), cellulose and cellulose derivatives such as cellulose acetate, cellulose nitrate, regenerated cellulose, nitrocellulose, celluloid or mixed cellulose esters (MCE).

13. A method according to any one of the preceding claims, wherein: The fluorescent compound can label mitochondria, cell membranes or nucleic acids.

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

15. A method for analyzing the microbial characteristics of a sample, comprising: - detecting the presence of at least one microorganism in said sample, - optionally, identifying said microorganism, - recovering said microorganism from said sample, and, - subjecting the recovered microorganism to a method according to any one of claims 1 to 14, wherein the method is used to determine the presence and / or extent of resistance of the microorganism to one or more antimicrobial agents, wherein the one or more antimicrobial agents are each at one or more concentrations.

16. The method according to claim 15, wherein: The sample is a biological fluid; Plant fluids, such as plant sap, nectar and root exudate; samples in culture medium or in bioreactors, such as cell cultures of higher eukaryotes, yeast, fungi or algae; liquids obtained from one (or more) animal or plant cells; liquid samples obtained from animal or plant tissues; liquid samples obtained from food matrices; liquid samples from chemical reactors; water from cities, rivers, ponds, lakes, oceans or air cooling towers; samples from liquid industrial effluents; in particular wastewater from intensive animal production or industry in the chemical, pharmaceutical, cosmetic or nuclear fields; liquid samples from pharmaceutical products; liquid samples from cosmetic products; fragrances; soil samples or mixtures thereof.

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