Method and device for detecting and optionally identifying microorganisms present on a surface arranged in the open air

Abstract

The invention relates to a method and device for detecting and optionally identifying microorganisms present on a surface arranged in the open air, consisting in directly targeting the volatile compounds resulting from the metabolic activity of the microorganisms present on said surface. The present invention also relates to such a method and such a device for determining the antimicrobial activity of a product.

Description

[0001] METHOD AND DEVICE FOR DETECTING AND POSSIBLY IDENTIFYING MICROORGANISMS PRESENT ON A SURFACE ARRANGED IN THE OPEN AIR

[0002] TECHNICAL FIELD

[0003] The present invention belongs to the field of gas sensors and in particular gas sensors useful for directly characterizing microorganisms present on an open surface, i.e. in the open air, by local detection, via one or more gas sensors, of the metabolic activity of these microorganisms.

[0004] The present invention provides a method and a device for detecting and possibly identifying microorganisms present on a surface placed in the open air.

[0005] STATE OF THE PRIOR ART

[0006] The microbiological analysis of a sample aims to qualitatively and / or quantitatively characterize the microorganisms present in the latter.

[0007] The time to obtain a result is limited by the minimum time required to obtain growth visible to the naked eye, for methods dependent on visual observation.

[0008] Optical methods using means such as, for example, magnifying lenses also require waiting for the formation of colonies or microcolonies, or other forms of growth visible using these means. They also depend on human intervention and are therefore not automatable.

[0009] However, automatable optical methods have been proposed.

[0010] Thus, patent EP 2376914B1 [1] describes a method and a device for the detection and identification of colonies and micro-colonies on the surface of a solid or semi-solid culture medium. The proposed method is based on the analysis of the fluorescence spectrum of the areas interrogated using a robotic microscopy device. However, the implementation of this method is long and tedious. The location of the colonies involves performing a discrete scan of the surface of the culture medium, by area of ​​100 pm in diameter, with 1 step of 100 pm. Each area is interrogated at different wavelengths. In addition, when the aim is not only to count, but also to identify the detected colonies, the method involves, after this first location step, a second step which consists of returning to the colonies to interrogate them individually, thus increasing the time taken to obtain results.

[0011] Perlemoine et al, 2021 [2] propose to detect, on the surface of an agar culture medium, phage lysis areas, using a wide-field microscopy device without a lens. Since the method described is based on the detection of growth zones, the time taken to obtain results is subject to the time required to obtain detectable growth. In addition, after seeding, the agar culture medium is placed on the device previously installed in an incubator. In fact, this process is not only cumbersome but also requires monopolizing the device for several hours for a single sample and therefore having as many devices as samples to be analyzed.

[0012] Other alternatives to visual observation involve computer-assisted image capture and processing devices and methods. However, one of the limitations of these computer-assisted optical methods is that they are not applicable to samples containing particles or when gas bubbles form in the culture medium, which can be confused with colonies or microcolonies and therefore lead to an erroneous result.

[0013] Another alternative to visual observation and advanced optical methods concerns the detection of volatile compounds emitted by microorganisms (or mCVs).

[0014] Patent application US 2008 / 0199904 A1 [3] describes a method based on the detection of volatile microbial metabolites, to detect, quantify and possibly identify microorganisms present in a cultured sample. To this end, the sensor used for the detection of volatile microbial metabolites is positioned inside the Petri dish containing a microbial culture on agar medium, so that the sensor is both positioned close to the surface of the agar medium and its sensitive part, i.e. reactive to volatile compounds, is visible from the outside. The sensor used is an inert paper-type support, on which are deposited in a localized manner, different chromogenic reagents capable of interacting with volatile microbial compounds. In this configuration, the sensor used therefore measures a mixture of volatile compounds emitted by all the microorganisms present on the surface of the culture medium.The method thus described therefore does not make it possible to distinguish, on the surface of a solid culture medium, different sources or different emission zones of volatile compounds, thus making it impossible to count these sources or these zones, their individual characterization and the measurement of the distances between these zones.

[0015] Such detection methods have attempted to be applied to the particular case of reading an antibiogram. International application WO 2013 / 163610 A1 [4] describes methods and devices for identifying and studying the antibiotic susceptibility of a cultured microorganism. This invention involves generating a headspace in contact with which a sensor sensitive to volatile compounds is placed, the colorimetric sensor being able to be a part of the container in which the headspace is generated. The determination of the antibiotic susceptibility of a microorganism as described in international application WO 2013 / 163610 A1 [4] is applied to the implementation of an antibiogram by dilution in a liquid medium but is not applicable to methods of antibiogram by diffusion in an agar medium in which the aim is to characterize the different growth zones on the surface of a culture medium.

[0016] Specific Diagnostics markets a rapid antibiogram method in the form of a kit and an automated reading device. This method involves the detection of mCVs emitted during the growth of a microorganism in the presence of an antibiotic. The kit consists of a microplate, each well of which contains a liquid culture medium, the microorganism to be studied, and a defined concentration of antibiotic. Detection is carried out using the microplate lid, which has a functionalized area sensitive to mCVs above each well. Thus, the antibiogram solution marketed by Specific Diagnostic is only applicable to antibiograms in liquid media.

[0017] Lim et al, 2014 [5] describe the detection and identification of microorganisms on the surface of a solid culture medium, based on the analysis of the emitted mCVs. To do this, the authors functionalize the lid of the Petri dish containing the sample to be analyzed, with a sensor in the form of a paper on which "spots" of different chromogenic reagents sensitive to mCVs have been deposited. In order to facilitate and automate the reading of the color code induced by the reaction of mCVs with the sensor reagents, the sensor is placed so that it can be read without removing the lid. Thus, the sensitive face of the sensor is not directly exposed to the surface of the culture medium but faces outwards. However, when at least two microbial species are present (case of polymicrobial samples), it becomes impossible to separate the mCVs emitted by each type of colony, thus making their identification impossible.

[0018] Green et al, 2011 [6] use an electronic nose for the identification of individual microbial colonies from an agar culture medium. The colonies to be identified are first collected and then suspended in a tube. The mCVs produced after closing the tube (after 300 seconds of incubation) are analyzed. Although this method allows individual characterization of colonies from a culture medium, it has the disadvantage of requiring several implementation steps: collection of the colony, suspension, generation of mCVs and then their analysis. Finally, the method described is not applicable to the measurement of growth inhibition diameter.

[0019] Reidt et al, 2020 [7] describe the use of an electronic nose coupled with a gas sampling device, for the analysis of mCVs emitted by microorganisms in culture on an agar nutrient medium. The device called "head sampler" is a container in which a microbial culture in a Petri dish is inserted without a lid. The analysis of the mCVs takes place after aspiration, towards the sensor, of the headspace generated inside the device. The analysis method thus described does not allow the surface of the medium to be characterized into different zones of mCV emission, all the mCVs emitted from the different zones of the surface mixing by diffusion in the headspace before being aspirated towards the sensor.

[0020] These solutions, since they implement the generation of a headspace, are not applicable to the format of the antibiogram by diffusion in agar medium, the configuration of which implies that the volatile compounds resulting from microbial metabolism and those spontaneously emitted by the culture medium diffuse and mix in this space, thereby losing the information on the location of their emission zones.

[0021] Olfactory mapping work has shown the possibility of detecting and locating sources of emissions of volatile compounds present on an open surface, i.e. exposed to the open air and therefore without resorting to the generation of a headspace, in particular by coupling an electronic nose type gas detector to a spatial displacement system [8]. Such a device would therefore make it possible to locate the zones of emissions of volatile compounds emitted, from the surface of an agar culture medium placed in the open air, in order to detect a zone of growth inhibition and possibly measure its diameter.

[0022] However, when the inventors used such a mapping device for measuring a growth inhibition zone on an agar culture medium containing microorganisms at confluence, the signals obtained do not allow the diameter of the inhibition zone to be measured, even though it is clearly visible and confluent. Indeed, the value of the diameter of the inhibition zone obtained using an olfactory mapping device differs from the value measured using optical means such as a “manual” measurement, using a caliper, resulting in poor characterization of the sensitivity of the microorganism to the antimicrobial agent tested.

[0023] The inventors therefore set themselves the goal of proposing a method and a device useful for detecting and possibly identifying microorganisms which do not have the limits and disadvantages of the methods and devices of the state of the art.

[0024] The inventors also set themselves the goal of proposing a method and a device useful for detecting and measuring the inhibitory effect of an antimicrobial agent on a microorganism with which it is brought into contact by diffusion, in particular in an agar medium, not having the limits and disadvantages of the methods and devices of the state of the art.

[0025] STATEMENT OF THE INVENTION

[0026] The solution proposed by the inventors consists of locally and directly targeting the volatile compounds resulting from the metabolic activity of the microorganisms present on a surface, thus making it possible to detect, locate, count and possibly identify them, more quickly than in the prior art since the present invention does not rely on the visualization of growth in the form of colonies or micro-colonies.

[0027] More particularly, the present invention relates to a method for detecting and possibly identifying microorganisms present on a surface placed in the open air, said method implementing a device comprising:

[0028] - a tube comprising a first open end and a second end fluidly connected to a measuring chamber;

[0029] - a measuring chamber comprising a gas-sensitive system designed to provide a signal representative of the presence of volatile compounds in a gaseous fluid; and

[0030] - suction means capable of sucking a gaseous fluid present at the first open end of the tube and bringing it from the first open end of the tube into the measuring chamber via the tube; said method comprising the following steps: a) positioning the first open end of the tube substantially vertically above a first zone ZI of said surface and at a distance less than or equal to 5R from this first zone ZI with R representing the desired spatial resolution, said tube having an internal diameter less than 2R, b) bringing the gaseous fluid present at the first zone ZI into the measuring chamber via said suction means, whereby, if a signal is provided by the system sensitive to the gas present in the measuring chamber, volatile compounds are present in the gaseous fluid brought into the measuring chamber,attesting to metabolic activity at the level of the first zone ZI and therefore the presence of microorganisms at the level of the first zone Zl, then c) position the first open end of the tube substantially vertically to a second zone Z2 of said surface different from the first zone Zl and at a distance less than or equal to 5R from this second zone Z2 and repeat step b) and possibly step c).,

[0031] More particularly, the present invention relates to a method for determining the antimicrobial activity of a product, said method implementing a device comprising a tube (6) comprising a first open end (61) and a second end (62) fluidically connected to a measuring chamber (4); a measuring chamber (4) comprising a gas-sensitive system designed to provide a signal representative of the presence of volatile compounds in a gaseous fluid; and suction means (5) capable of sucking a gaseous fluid present at the first open end of the tube and bringing it from the first open end of the tube into the measuring chamber via the tube; said method comprising the following steps:

[0032] - seed a surface with microorganisms,

[0033] - bringing the surface into contact with a solution or a solid support comprising at least one product exhibiting antimicrobial activity,

[0034] - continuously scanning the surface placed in the open air, before the microbial culture is visible to the naked eye, according to the following sub-steps: a) positioning the first open end of the tube substantially vertically to said surface and at a distance less than or equal to 5R from this surface with R representing the desired spatial resolution, said tube having an internal diameter less than 2R, b) bringing the gaseous fluid present at the surface into the measuring chamber via said suction means, whereby the system sensitive to the gas present in the measuring chamber provides signals characterizing the volatile compounds present in the gaseous fluid brought into the measuring chamber and, from the signals provided, the zone of inhibition of the microorganisms by said at least one product having antimicrobial activity is determined.

[0035] In other words, when the method according to the invention is a method for determining the antimicrobial activity of a product, the inventors propose to “scan”, after an incubation time less than the time necessary to obtain clearly visible and confluent microbial growth, the surface of an agar culture medium using an olfactory mapping device to detect and locate,

[0036] - not only volatile compounds, in particular amino compounds, resulting from microbial metabolism, making it possible to locate growth zones delimiting a growth inhibition zone, or

[0037] - but also, and very surprisingly, volatile compounds, particularly amino acids, spontaneously emitted by the culture medium and whose emission is modulated by the presence of metabolically active microorganisms.

[0038] This detection and localization makes it possible to measure exactly the diameter of a zone of microbial growth inhibition.

[0039] Prior to the present invention, the use of an olfactory mapping device for measuring zones of microbial growth inhibition from a culture medium, particularly agar, had never been described and, moreover, by targeting not the volatile compounds emitted by the microorganisms but the volatile compounds spontaneously emitted by the culture medium.

[0040] Furthermore, contrary to what is expected, it is by placing oneself in conditions of low microbial growth and therefore in conditions for which the concentration of volatile compounds produced is the lowest, that the measurement of a growth inhibition zone, from a surface permeable to volatile compounds, is obtained with accuracy, that is to say with a deviation from the true value less than or equal to 1 mm.

[0041] Furthermore, in addition to making it possible to automate and robustify the measurement of the diameter of a growth inhibition zone, by placing it in conditions of low microbial growth, the proposed solution provides the advantage of reducing by several hours the time required to obtain a sensitivity result to an antimicrobial agent (i.e. from more than 16 hours to less than 6 hours).

[0042] In addition, the inventors propose to "scan" the surface exposed to the open air such as an agar culture medium after opening the Petri dish or any other container in order to eliminate the naturally formed headspace.

[0043] The term "headspace" refers to a gas in which the various volatile compounds emitted by different sources present on this surface diffuse, concentrate and mix. In other words, the formation of a headspace corresponds to the accumulation of volatile compounds in a closed space. This headspace can be used to increase their concentration during the secretion process. The headspace can therefore be defined as a volume of gas located between the surface of a liquid or solid sample contained in a container and the means of closing this container such as a lid. The headspace corresponds, for example, to the volume of gas located between the product and the seal of the packaging containing this product.

[0044] In the context of the present invention, this headspace is no longer considered as the sample to be analyzed, but is considered as noise, an interference to be eliminated before implementing the method.

[0045] The inventors have shown that using a headspace as in the methods of the prior art is incompatible with solving the problems and limitations of the latter. Indeed, the generation of a headspace aims to concentrate microbial volatile compounds (mVCs) emitted by the microorganisms in a volume of gas contained between the surface of the culture medium and the walls of the container (including the lid or cap) to thus achieve mVC concentrations sufficient to be analyzed. However, a headspace containing such concentrations is generated after a certain period of time, which lengthens the time taken to obtain results.

[0046] Furthermore, the analysis of mCVs from a headspace is disadvantageous due to a diffusion and mixing effect of the latter, making it impossible to distinguish the different emission zones existing on the surface. Indeed, in the headspace of a Petri dish or any other container of a culture medium, the mCVs emitted by colonies belonging to different species diffuse and mix, thus making their identification impossible (impossibility of linking particular volatile compounds to a particular type of colony) and their precise localization.

[0047] Another disadvantage of using a headspace is the impossibility of determining the antibiotic resistance of a microorganism when carried out by the agar diffusion method. Indeed, in this type of test, the headspace necessarily contains mCVs emitted by the growing microorganisms and delimiting a zone of inhibition. However, it is impossible to estimate the diameter of an inhibition zone by analyzing the mCVs contained in a headspace.

[0048] Thus, the present invention avoids the mixing of volatile compounds and makes it possible to transcribe the true heterogeneity of emission into volatile compounds of a culture medium on which microorganisms are present. The method according to the present invention therefore has the originality of being free from the constraint of generating a headspace or waiting for the formation of colonies visible to the naked eye or other distinct growth zones. It gains in precision, broadens the type of samples that can be analyzed, and facilitates the obtaining of rapid results.

[0049] One of the essential characteristics of the method according to the present invention is its implementation on a surface placed in the open air.

[0050] By "surface exposed to the open air" is meant an open surface, i.e. a surface whose overhanging gas volume is not contained in a closed space and therefore, the volatile compounds emanating from this surface do not form a headspace. A surface exposed to the open air therefore corresponds to a surface of a sample above which a headspace cannot form due to the absence of any means enclosing this surface in a closed space. A surface exposed to the open air also includes the fact that the surface may be in the environment of a closed enclosure such as an incubator, an automaton or other but whose volume does not allow the formation of a headspace, i.e. a volume in which the volatile compounds are concentrated. In addition, in such an enclosure, the atmosphere may be air, an inert gas such as nitrogen or any other gas mixture without oxygen (case of anaerobic microorganisms).

[0051] Any surface on which microorganisms may be present or are likely to develop in depth may be used within the scope of the present invention. The presence of microorganisms on the surface used in the context of the method according to the invention may be intentional, as in the case of seeding. On the contrary, the presence of microorganisms on the surface used in the context of the method according to the invention may be the consequence of a particular event such as contamination, infection, pathology or malfunction of a decontamination or cleaning system.

[0052] Advantageously, the surface used in the context of the method according to the invention is chosen from the group consisting of a surface of a solid or semi-solid support, a surface of a human or animal body such as a wound, an agar culture medium and a filtration membrane.

[0053] When the method according to the invention is a method for determining the antimicrobial activity of a product, the surface used is an agar culture medium. Examples of surfaces of a solid support include the surface of a large installation such as an industrial object such as a worktop, an electronic device or a machine used in the food, pharmaceutical or cosmetic industries, a tank, a restaurant kitchen, a cold room, a sanitary facility, a container or the surface of a small object such as medical devices or pipes. It may thus be a surface in contact with food, sterile objects or objects whose microbial load must be controlled.

[0054] For such surfaces, it is possible, prior to implementing the method according to the invention, to bring these surfaces into contact with a solution containing compounds neutralizing disinfectant substances such as, for example, Tween® 80 or sodium thiosulfate and / or precursor compounds of volatile microbial compounds.

[0055] In a particular embodiment, the method according to the present invention may comprise a prior step of seeding the surface with microorganisms.

[0056] Furthermore, when the method according to the invention is used in particular to study the antimicrobial activity of a product as explained below, the method comprises a prior step of bringing the surface into contact with a solution or a solid support comprising at least one product capable of exhibiting antimicrobial activity.

[0057] When the surface used is an agar culture medium, it may be the surface of a culture medium contained in a container such as a Petri dish and the lid of which has been removed prior to implementing the method according to the invention in order to expose the surface to the open air and thus eliminate the head space initially contained in the closed container.

[0058] In this embodiment, the agar culture medium may be solid or semi-solid and the microorganisms to be characterized have been, prior to bringing the surface into contact with a solution or a solid support comprising at least one product capable of exhibiting antimicrobial activity, seeded on the surface or in depth.

[0059] Alternatively, it is possible to contact the surface with a solution or a solid support comprising at least one product capable of exhibiting antimicrobial activity without prior seeding.

[0060] The term "seeding" is used in its strict microbiological definition, that is, the deliberate introduction of microorganisms into a culture medium to promote their metabolic activity, growth, and study. However, in some cases of use, the surface placed in the open air already has microorganisms. However, in the context of this invention, its definition is broadened to non-deliberate seeding, which corresponds to an involuntary introduction or presence of microorganisms in a medium conducive to their metabolic activity. This broadened definition therefore includes cases of food contamination and infections of human or animal wounds.

[0061] In another embodiment, the culture medium may be liquid and, in this case, the surface to be scanned is an inert support such as, in a non-limiting manner, a filtration membrane, one of the faces of which is in contact with the liquid culture medium and the other face of which, in contact with a gas, contains the microorganisms to be characterized.

[0062] Among the microorganisms that one wishes to detect and possibly identify by the method according to the invention, one finds both prokaryotic microorganisms such as archaea or Gram-positive or Gram-negative bacteria, as well as eukaryotic microorganisms such as yeasts and other microscopic fungi and protists such as algae or protozoa.

[0063] As illustrative and non-limiting examples of microorganisms present or likely to be present on the surface used in the context of the invention, mention may be made of bacteria of the Enterobacteriaceae family, bacteria of the Pseudomonadaceae family, bacteria of the Staphylococcus genus, bacteria of the Streptococcus genus, bacteria of the Campylobacter genus, bacteria of the Haemophilus genus, bacteria of the Anaerococcus genus, bacteria of the Bacteroides genus, bacteria of the Acinetobacter genus, bacteria of the Stenotrophomonas genus, bacteria of the Achromobacter genus, bacteria of the Fusobacterium genus, bacteria of the Pasteurella genus, bacteria of the Bacillus genus, bacteria of the Listeria genus, bacteria of the Clostridium genus, bacteria of the Mycobacteria genus, bacteria of the Enterococcus genus and bacteria of the Pasteurella genus, yeasts of the Candida genus, yeasts of the Saccharomyces,fungi of the genus Aspergillus, fungi of the genus Penicillium and / or archaea of ​​the genus Methanobrevibacter.,

[0064] In addition, among the bacteria of the Enterobacteriaceae family present or likely to be present on the surface used in the context of the invention, mention may be made of bacteria of the genera Escherichia, Salmonella, Shigella, Enterobacter, Klebsiella, Serratia, Proteus, Morganella, Yersinia, Citrobacter and Providencia. The scope of application of the method according to the invention has the advantage of covering monomicrobial samples, but also polymicrobial samples.

[0065] Advantageously, in the context of the method according to the present invention, the microorganisms to be detected and possibly identified are in the form of a colony, a micro-colony, a confluent microbial mat or a set of isolated microbial cells, visible or not to the naked eye.

[0066] When the method according to the present invention is a method for determining the antimicrobial activity of a product, the microorganisms to be detected and possibly identified are in the form of a colony, a micro-colony, or a set of isolated microbial cells, visible or not to the naked eye.

[0067] As previously explained, it is not necessary for the microorganisms that one seeks to detect and possibly identify by the method according to the invention to be visible to the naked eye.

[0068] Indeed, one of the advantages of the method according to the invention is the obtaining of a detection and a localization of the metabolic activity of a microbial culture visible to the naked eye, but also a microbial culture not visible to the naked eye, as long as the incubation period has been sufficient for the microorganisms to enter a phase of metabolic activity. This incubation period is however insufficient to obtain a carpet of confluent and clearly visible colonies.

[0069] By "metabolic activity of microorganisms" we mean a set of chemical and / or biochemical reactions which take place within the microbial cell or outside of it (case of excreted enzymes) and which generate at least one volatile compound.

[0070] Volatile compounds designate a set of organic and inorganic molecules which have a vapor pressure greater than or equal to 0.01 KPa at a temperature of 293.15 K, or having a corresponding volatility under particular conditions of use. Preferably, the organic and inorganic molecules have a vapor pressure greater than or equal to 0.0001 Pa at the temperature at which the process is carried out. As illustrative and non-limiting examples of microbial volatile compounds, mention may be made of sulfur compounds, such as hydrogen sulfide, dimethyl sulfide, dimethyl disulfide, methyl mercaptan; amine and diamine compounds such as ammonia, putrescine, cadaverine, monomethylamine, dimethylamine, ethylamine, trimethylamine, isopropylamine, methylethylamine, dimethylethylamine, diethylamine, methyldiethylamine;short-chain organic acids such as those between acetic acid and capric acid and their methylated derivatives such as isovaleric acid and volatile esters such as ethyl acetate and ethyl butanoate; unbranched short-chain aliphatic compounds such as 1,3-butanediene and branched aliphatic compounds such as 2,4-dimethyl-1-heptane, 1-undecene, isoprene; aromatic cyclic compounds such as 2-aminoacetophenone, benzonitrile, toluene, formaldehyde and alicyclic compounds such as indole, skatole, 4-methyl quinazoline; and carbonyl compounds such as 2-methylbutanal, 3-hydroxybutanone, acetaldehyde, formaldehyde, 2-butanone, 2-pentanone, 2-heptanone and 2-nonanone.;

[0071] Consequently, prior to implementing the method according to the present invention, it may be necessary to subject the microorganisms that one wishes to detect and possibly identify, to an incubation period, in the presence of compounds capable of leading to the emission of at least one volatile compound by metabolic pathway, whether they are of a nature to promote cell multiplication (case of culture media) or not. These compounds may be naturally present in the medium or added extemporaneously.

[0072] In a particular embodiment, in particular when the method according to the invention is a method for determining the antimicrobial activity of a product, it is the volatile compounds spontaneously emitted by the surface, i.e. the agar culture medium, and the emission of which is modulated by the microbial metabolic activity, which are targeted to detect and locate zones of microbial growth delimiting zones of growth inhibition. In this embodiment, the detection of the microbial metabolic activity results not from an emission of volatile compounds but from a reduction in the emission of volatile compounds which is measured. As an illustrative and non-limiting example, the spontaneous emission of ammonia by an agar culture medium can be reduced by microorganisms capable of assimilating, by metabolic pathway, ammonium as a source of nitrogen. This particular embodiment is notably illustrated in example 4.

[0073] In this embodiment, when it is the volatile compounds spontaneously emitted by the surface, i.e. the agar culture medium, and whose emission is modulated by microbial metabolic activity, which are present in the gaseous fluid brought into the measuring chamber, we observe

[0074] - a decrease in the signals provided by the gas-sensitive system of the measuring chamber when the first open end of the device tube passes from an inhibition zone to a microbial growth zone, or

[0075] - an increase in the signals provided by the gas-sensitive system of the measuring chamber when the first open end of the device tube passes from a microbial growth zone to an inhibition zone.

[0076] Alternatively, in particular when the method according to the invention is a method for determining the antimicrobial activity of a product, it is the volatile compounds emitted by the microbial metabolic activity which are targeted to detect and locate zones of microbial growth delimiting zones of growth inhibition. In this embodiment, the detection of the microbial metabolic activity results from an emission of volatile compounds which is measured. This particular embodiment is notably illustrated in example 3.

[0077] In this embodiment, when it is the volatile compounds emitted by microbial metabolic activity which are present in the gaseous fluid brought into the measuring chamber, we observe

[0078] - an increase in the signals provided by the gas-sensitive system of the measuring chamber when the first open end of the device tube passes from an inhibition zone to a microbial growth zone, or

[0079] - a decrease in the signals provided by the gas-sensitive system of the measuring chamber when the first open end of the tube of the device passes from a zone of microbial growth to a zone of inhibition. Without inventive effort, the person skilled in the art knowing the zone(s) of application of the antimicrobial product(s) will be able to determine, during the continuous scanning of the surface, the embodiment concerned.

[0080] It is possible to represent in graphic form the signals generated by the gas-sensitive system of the measuring chamber as a function of the position of the first open end of the suction tube. In such a graphical representation, the transition from a microbial growth zone to an inhibition zone or from an inhibition zone to a microbial growth zone is materialized on the graph by a change in slope.

[0081] Another of the essential characteristics of the method according to the present invention is the particular device used for its implementation. This device comprises (i) a tube comprising a first open end and a second end fluidly connected to a measuring chamber; (ii) this measuring chamber comprising a gas-sensitive system designed to provide a signal representative of the presence of volatile compounds in a gaseous fluid; and (iii) suction means such as a pump, capable of sucking a gaseous fluid present at the first open end of the tube and bringing it from the first open end of the tube into the measuring chamber via the tube.

[0082] The suction tube that the device has in the present invention can be made of any material that does not interact with the volatile compounds intended to be conveyed by this tube. By way of illustrative and non-limiting example, this tube can be made of polytetrafluoroethylene (PTFE).

[0083] In the context of the present invention, the detection, localization, counting and identification of microorganisms present on a surface are therefore carried out by measuring, from this surface placed in the open air, volatile compounds resulting from microbial metabolism, directly above the emission zone. By this direct and local measurement, the information generated on the state of microbial metabolism is almost in real time, without intermediate storage of volatile compounds (headspace, adsorption on resin or carbon, etc.). The direct and local measurement of the emission of volatile compounds is possible by the proximity, direct or indirect, of the measuring chamber with the zone to be characterized. This proximity of the measuring chamber with the surface of the zone to be characterized prevents the diffusion and dilution of the volatile compounds emitted from the zone in the surrounding gas volume.

[0084] To enable such measurement conditions, it is necessary to carry out an adequate selection of several parameters regarding the device implemented.

[0085] Thus, the internal diameter of the suction tube (D) must be precisely selected. Indeed, its value conditions the desired spatial resolution (R) which corresponds to the resolution of the scan, i.e. the capacity to measure a signal variation between 2 different sources of volatile compounds. For example, if the internal diameter of the tube is 1 mm, the method described will be able to measure a signal variation between 2 microbial colonies at least 1 mm apart. If the distance between these 2 colonies is 0.5 mm, the measured signal will result from a mixture between the volatile compounds coming from these 2 colonies. Consequently, D is strictly less than 2R and advantageously D is less than or equal to R.

[0086] For example, the internal diameter of the suction tube is between 0.2 mm and 2 mm. Advantageously, the internal diameter of the suction tube is between 0.5 mm and 1 mm. This configuration is particularly suitable when the surface characterization must be precise, such as, for example, in the case of measuring the diameter of a zone of growth inhibition or metabolic activity.

[0087] In a particular embodiment, the open end of the suction tube positioned close to the surface to be scanned can be fluidically connected to an inverted funnel, i.e. with a larger diameter on the surface side, typically this diameter is between 20 mm and 150 mm. In particular, this embodiment is suitable for use on large surfaces such as previously defined as worktops.

[0088] Similarly, the distance (d) between the first open end of the tube and the emission surface, i.e. the surface to be scanned and in particular the zones Z1 and Z2 previously described, is a function of the desired spatial resolution (R) in order to avoid dilution and diffusion of volatile compounds in the ambient air. Thus, the distance (d) between the first open end of the tube and the surface is less than or equal to 5R and preferably d is less than or equal to 2R.

[0089] Furthermore, the linear velocity of the gaseous fluid entering the suction tube must be sufficient to convey the volatile compounds to the measuring chamber during the scan, without introducing pollution by the emission zones near the targeted zone, i.e. the zone ZI during step b) of the process, nor diluting the volatile compounds by the ambient air. Consequently, the linear velocity of the gaseous fluid in the tube is typically between 0.01 m / s and 10 m / s and advantageously between 0.1 m / s and 5 m / s.

[0090] Finally, the spatial sampling frequency (FS) corresponds to the number of measurements per unit of space. It is directly related to the desired spatial resolution R. This quantity allows the signal to be reconstructed or not. The spatial sampling frequency (FS) is at least 1 / R.

[0091] It may be desirable for the spatial sampling frequency (SRF) to be as high as possible. For example, with a series of colonies of diameter X and all spaced a distance of Y, the spatial pattern has a period of (X+Y) and therefore a frequency of 1 / (X+Y). This corresponds to having at least one measurement per spatial unit of resolution, regardless of the speed or number of scans.

[0092] The gas-sensitive system presented by the measuring chamber of the device as implemented in the method according to the invention comprises at least one gas sensor.

[0093] In a particular embodiment, this gas sensitive system comprises only one gas sensor.

[0094] In another particular embodiment, this gas sensitive system comprises a plurality of gas sensors. In this embodiment, the sensors may be placed in parallel. Alternatively, the sensors may be placed in series, i.e., they are fluidically connected to each other. Alternatively, a portion of the sensors may be placed in parallel and the remaining portion in series.

[0095] Any type of gas sensor can be used within the scope of the present invention.Thus, the gas sensor(s) that comprise the system sensitive to the gas present in the measuring chamber of the device used during the method according to the invention is / are chosen from the group consisting of spectrophotometric gas sensors (Infrared (IR) or UltraViolet (UV)), photoionizing gas sensors (PID), spectrometric gas sensors such as spectrometric gas sensors using selected ion flow tube mass spectrometry (or SIFT-MS for "Selected Ion Flow Tube-Mass Spectrometry") or proton transfer reaction mass spectrometry (or PTR-MS for "Proton-Transfer-Reaction Mass Spectrometry"), electrochemical gas sensors, metal oxide semiconductor gas sensors and sensors based on molecular affinity recognition, whether the transduction mode is optical or gravimetric.

[0096] In a particular embodiment, the gas-sensitive system present in the measuring chamber of the device used during the method according to the invention may belong to the group of "electronic noses". The electronic nose owes its name to the analogy that exists between its operation and that of the human olfactory system. An electronic nose is a device that uses several sensors to detect volatile compounds. These sensors may be electrochemical, photonic, gravimetric, metal oxide, conductive polymer or any other type described in the state of the art. The electronic nose also comprises a computer system ensuring the processing of the responses emitted by the sensors in the form of signals, this system playing the role of the human brain.

[0097] Advantageously, the gas sensor(s) that comprises the system sensitive to the gas present in the measuring chamber of the device used during the method according to the invention is one or more optical sensors such as interferometric sensors or plasmonic sensors.

[0098] It is obvious that the response time of the gas sensor(s) included in the gas-sensitive system present in the measuring chamber of the device used must be compatible with the resolution of the desired scan. For example, for scanning the surface of an agar culture medium with a resolution of 1 mm and a movement speed of 5 mm / s, the minimum response time of the sensor used must be 200 ms. The movement speed for performing the scan can also be adapted according to the response time of the sensor.

[0099] In a particular embodiment, the gas-sensitive system further comprises at least one humidity sensor. Any type of humidity sensor can be used in the context of the present invention and in particular any type of commercially available humidity sensor. Advantageously, the humidity sensor used will have a response time similar to the response time of the volatile compound sensor implemented in the method. The presence of a humidity sensor makes it possible to correct the signal measured by the gas sensor according to the humidity present. This characteristic is particularly advantageous for use on a wet surface such as a culture medium or a human or animal wound.

[0100] Advantageously, this humidity sensor is placed in series after the gas sensor or is one of the gas sensors that comprise the gas-sensitive system present in the measuring chamber of the device used during the method according to the invention.

[0101] In the method according to the invention, it is possible to scan the surface several times at regular or irregular intervals. In other words, in the method according to the invention, the first open end of the suction tube can be positioned substantially vertically above the same area of ​​the surface during two separate steps c). It is obvious that these two separate steps c) are not consecutive. This variant of the method according to the invention makes it possible to follow the temporal evolution of the microbial metabolism at the level of the same area of ​​the surface or even at the level of the entire surface. As specific examples, it is possible to envisage scanning an area of ​​the surface or the entire surface once per hour.

[0102] In a first particular embodiment, the passage from the first zone Z1 to a second zone Z2 of the surface during step c) or between two consecutive zones of this surface during a repetition of steps b) and c) is automated. Such an automated passage typically implements a robotic system. In a second particular embodiment, the passage from the first zone

[0103] ZI to a second zone Z2 of the surface during step c) or between two consecutive zones of this surface during a repetition of steps b) and c) is manual.

[0104] Whether the passage from the first zone ZI to a second zone Z2 of the surface during step c) or between two consecutive zones of this surface during a repetition of steps b) and c) is robotic or manual, the following different forms of implementation can be considered.

[0105] When the method according to the invention is a method for determining the antimicrobial activity of a product, the scanned areas do not advantageously represent the entire surface of the culture medium; scanning only the areas located on a linear, rectilinear or curved trajectory and passing through the application area and in particular through the center of the initial deposit of the antimicrobial agent may be sufficient to measure the diameter of the inhibition zone centered around this deposit. In all cases, the scan must be adapted to the spatial configuration of the deposits of antimicrobial agents on the surface.

[0106] In a first embodiment, the open end of the suction tube is held in a fixed position and it is the surface which is moved so that the area opposite the open end of the tube passes from zone Z1 to zone Z2 or from zone Zn to zone Zn+1. This first embodiment when the method according to the invention is a method for determining the antimicrobial activity of a product consists of scanning the surface, the latter following a rotational movement, in particular when placed on a rotating plate. During this rotation, the application zone(s) of the antimicrobial product(s) applied to the surface is / are brought vertically above the open end of the tube held fixed.

[0107] In a second embodiment, the surface is kept fixed and it is the open end of the suction tube that is moved from zone ZI to zone Z2 or from a zone Zn to zone Zn+1. This second embodiment when the method according to the invention is a method for determining the antimicrobial activity of a product consists of scanning the surface such as an agar culture medium with the open end of the tube following a rectilinear linear trajectory passing through the application zone(s) of the antimicrobial product(s) applied to the surface. In a third embodiment, the open end of the suction tube and the surface are both moved so that the area opposite the open end of the tube moves from zone ZI to zone Z2 or from a zone Zn to zone Zn+1. In this third embodiment, the movements can be made sequentially or simultaneously.In a particular configuration of this form of implementation, the open end of the tube follows a longitudinal displacement along an axis and the surface such as a culture medium follows a rotational movement, in particular when placed on a rotating plate.

[0108] Note that when the open end of the suction tube is moved, the measuring chamber can follow the same movement or, on the contrary, remain in a fixed position. The choice between these two alternatives depends essentially on the size of the surface to be scanned.

[0109] The surface scan is continuous, but not necessarily at constant speed. In other words, the speed of movement between zone Z1 and zone Z2 and between two consecutive zones Zn and Zn+1 can be constant or, on the contrary, variable.

[0110] In the case of a surface of the culture medium type in a Petri dish, the diameter of the area interrogated is typically 1 mm and the duration of a scan of the entire surface is less than 10 min. Finally, with the solution proposed by the method according to the invention, if the gas sensor implemented allows microbial identification, then the identification of the detected colonies is done during the detection scan without this generating a negative impact on the duration of the scan.

[0111] Finally, in certain embodiments of the method according to the invention, at least one of the scanned zones, i.e. one of the zones vertically above which the open end of the tube of the device used is positioned and from which the gaseous fluid is brought into the measuring chamber, may be a reference zone such as a positive control (zone in which there are microorganisms) or a negative control (zone free of microorganisms).

[0112] The present invention also relates to a device for implementing a method as previously defined. This device comprises - a tube comprising a first open end and a second end fluidly connected to a measuring chamber, said tube being as previously defined;

[0113] - a measuring chamber comprising a gas-sensitive system designed to provide a signal representative of the presence of volatile compounds in a gaseous fluid, said gas-sensitive system being as previously defined, said gas-sensitive system possibly comprising at least one humidity sensor;

[0114] - suction means capable of sucking a gaseous fluid present at the first open end of the tube and bringing it from the first open end of the tube into the measuring chamber via the tube; and

[0115] - means capable of moving said first open end of the tube and / or a surface to be analyzed, i.e. a surface presenting or likely to present microorganisms.

[0116] In a particular embodiment, the device according to the invention may further comprise means capable of locating this device and / or the first open end of the tube in space. As particular examples of such locating means, mention may be made of stepper motors, linear motion sensors, rotary motion sensors, absolute position sensors and relative position sensors.

[0117] In addition, the device according to the invention may also comprise

[0118] - any other element allowing dilution in the event of excessively strong signals, such as, for example, a dilution chamber, and / or

[0119] - any other element allowing the filtration of volatile compounds in order to modify the composition of the gas analyzed, such as, for example, Nafion tubing, and / or

[0120] - connectors to ensure the watertightness of the connections between the different elements making up the device.

[0121] The present invention also relates to the use of a method as previously defined or of a device as previously defined for: - determining the contamination of a surface by microorganisms, or

[0122] - count the microorganisms on a surface, or

[0123] - identify microorganisms on a surface, or

[0124] - determine the antimicrobial activity of a product, or

[0125] - monitor over time the metabolic activity of microorganisms present on a surface.

[0126] As regards the determination of the contamination of a surface by microorganisms, this use finds applications in the food industry and / or in the pharmaceutical field.

[0127] Determining the contamination of a surface by microorganisms consists not only of detecting these microorganisms by determining the presence or absence of one or more mCV emission zones on the surface to be scanned, but also of locating these microorganisms by identifying in the space represented by the surface to be scanned, one or more mCV emission zones.

[0128] Thus, when the method or device according to the invention is used to determine the contamination of a surface by microorganisms, this use can, in certain cases, be implemented to calculate distances between the zones which emit different volatile compounds, these distances being able to be a growth inhibition diameter, a metabolic inhibition diameter or a lysis range.

[0129] With respect to the counting of microorganisms on a surface, this use may consist of counting areas of growth or areas of metabolic activity on the whole of the surface or on a part of it.

[0130] Regarding the identification of microorganisms on a surface, this use consists of differentiating and identifying microorganisms individually on the basis of the volatile compounds they emit. Note that a differentiation of microorganisms into different groups, based on their differences in qualitative or quantitative emissions of volatile compounds, does not necessarily lead to identification. The identification of microorganisms aims to determine the belonging of a microorganism to a taxon. These taxa can be those commonly accepted in microbial taxonomy (family, genus, species, etc.) or correspond to a group defined by one or more metabolic characteristics (coliforms, sulfite-reducing anaerobes, etc.) or to a group characterized by its resistance to one or more inhibitory molecules (MRSA: Methicillin Resistant Staphylococcus Aureus, etc.).This use to identify microorganisms may, in certain forms of implementation, require a comparison step with the volatile compounds emitted by known microorganisms.

[0131] With regard to the study or determination of the antimicrobial activity of a product, the latter may be a chemical compound such as a disinfectant, a biochemical compound such as an antimicrobial peptide or an antibiotic, or an active biological compound such as a phage or a composition containing at least one of these compounds such as a phage preparation or a preparation containing several compounds with antimicrobial activity. The antimicrobial activity exhibited by this product may be an antibacterial activity, an antifungal activity or an antiviral activity. In a particular form of implementation, this use may consist of studying the antibiotic susceptibility of microorganisms.

[0132] With regard to the monitoring over time of the metabolic activity of microorganisms present on a surface, this use consists of carrying out scans at different times to obtain a temporal comparison in order to monitor the evolution of the metabolism of the microorganisms present on the scanned surface or on a given area of ​​the latter.

[0133] Other characteristics and advantages of the present invention will become apparent to those skilled in the art upon reading the examples below given for illustrative and non-limiting purposes, with reference to the appended figures.

[0134] BRIEF DESCRIPTION OF THE DRAWINGS

[0135] Figure 1 is a schematic representation of a device according to the invention.

[0136] Figure 2 shows the time evolution of the signals measured by the NOA mCVs sensor during the TSA agar scan. The arrows indicate the intensity peaks corresponding to the passage of the sensor tubing over a colony. Tl

[0137] Figure 3A shows the signature obtained from a colony belonging to the species S. aureus (after L2 normalization).

[0138] Figure 3B shows the signature obtained from a colony belonging to the species E. coli (after L2 normalization).

[0139] Figure 4 shows a photograph of Mueller Hinton culture medium after 4 h of incubation at 37°C. The antibiotic discs from left to right are SXT 25 and GME 30. The dotted arrow indicates the direction of movement of the sensor tubing. The dotted circles inscribed between the arrows indicating the positions 7 mm, 33 mm, 47 mm and 65 mm, symbolize the zones of metabolic inhibition detected by the NOA after 4 h of incubation.

[0140] Figure 5 shows the evolution of the average intensity of the signals measured by the mCVs sensor as a function of the distance traveled by the sensor tubing along the diameter of the culture medium.

[0141] Figure 6 shows a photograph of the measuring device implemented in example 4, to evaluate the antibiotic susceptibility of a strain of S.aureus (ATCC 25923) after 6 hours of incubation with 1: Mueller-Hinton E agar; 2: Antibiotic disc; 3: Rotating plate; 4: Stand; 41: Height-adjustable stand arm; 42: Height-adjusting screw of the stand arm; 5: Suction tube; 51: Open end of the suction tube; 6: Stepper motor; 7: NeOse Advance electronic nose housing comprising a volatile compound suction pump, a fluid line connecting the pump to the measuring chamber and an electronic system for controlling the pump and the volatile compound sensor; 8: Measuring chamber of the NeOse Advance volatile compound sensor; 9: Data processing computer system; 91: Measured signal for sensors sensitive to amino volatile compounds; 92: Signal I measured for sensors not sensitive to amino volatile compounds.

[0142] Figure 7 shows the evolution of the measured signal intensity for the sensors sensitive to amino volatile compounds of the NOA volatile compound sensor as a function of the position of the analyzed zone. The dotted lines materialize the start and end positions of the inhibition zones for the antibiotic discs SXT25 (SXT), AMC3 (AMC), CN30 (CN) and RD5 (RD). DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

[0143] Example 1: General principle and device according to the invention

[0144] An agar culture medium 1, the lid of which has been removed to eliminate the headspace, is placed on a rotating plate 3 forming part of an automated system for measuring zones of inhibition 10.

[0145] Culture medium 1 was inoculated with a bacterial strain in order to determine its sensitivity to different antibiotic discs 2.

[0146] The measurement of the inhibition zones is carried out via a scan of the surface of the culture medium 1 aimed at detecting volatile compounds including microbial volatile compounds emitted from this surface.

[0147] The scan is carried out by moving along an axis 8 the end 61 of the suction tube 6 positioned above the culture medium 1 and inserted through a movable element 7. A rotation of the rotating plate 3 allows the scanning of the different inhibition zones.

[0148] The CVs and in particular the mCVs are sucked by the action of a pump 5 via a suction tube 6, towards a measuring chamber 4 comprising a CV sensor and in particular a mCV sensor, the end 62 of the suction tube 6 being connected to the measuring chamber 4.

[0149] The collected data is analyzed by a data processing system 9.

[0150] Example 2: Counting and identifying colonies by olfactory scanning of the surface of a culture medium.

[0151] Tests were performed to count and identify colonies present on the surface of TSA agar.

[0152] After inoculation by spreading 50 pL of a tryptone-salt solution (VWR, AX011130) containing bacteria belonging to the species S. aureus (ATCC 25923) and E. coli (ATCC 11775), the TSA agar (VWR, 301114ZA) is incubated for 18 hours at 35°C. After incubation, the lid of the Petri dish is removed and the TSA agar is placed on the fixed platform of a robotic X, Y axis (Figure 1). A NeOse Advance (NOA) electronic nose (Aryballe) is fixed on the mobile part of this axis. A PTFE tube (1 mm internal diameter), connected to the sensor at one end and fixed to a support secured to the moving part of the axis at the other end, is positioned at the edge of the agar to be scanned, at a distance of 2 mm above the surface. The NOA pump located upstream of the sensor is programmed to have a constant suction flow rate of 30 mL / min. Signal acquisition is started at the same time as the automated movement of the moving part of the axis.The movement is programmed to scan the entire surface in approximately 180 seconds.

[0153] The temporal evolution of the intensity of the signals measured by the electronic nose when moving the suction tube over the culture medium is presented in the form of a graph (Figure 2). Each intensity peak indicated by an arrow on the graph corresponds to the passage of the suction tube over a colony. The increase in the intensity of the signals measured when the tubing passes over a colony corresponds to the detection of mCVs specifically emitted by this colony. Thus, the 22 peaks identified reflect the presence of 22 colonies on the surface of the scanned agar. Finally, the analysis of the signature for each emission peak in mCVs (Table 1) reveals 2 types of signatures as presented respectively in Figure 3A and Figure 3B corresponding to the 2 species present on the surface of the agar.

[0154] Table 1: Distribution of the different peaks according to their belonging to a signature type.

[0155] In conclusion, the olfactory scan of the surface of the culture medium, without resorting to the analysis of a headspace, made it possible to count and identify 13 colonies of S. aureus and 9 colonies of E. coli in a rapid and automated manner. Example 3: Measurement of inhibition diameters for the evaluation of the antibiotic susceptibility of a microorganism by the local detection of mCVs emitted on the surface of an agar culture medium.

[0156] The antibiotic susceptibility of the E. coli strain (ATCC 11775) is evaluated using the agar diffusion method.

[0157] After inoculation on Mueller-Hinton agar (VWR, OXOIPO5139A), by swabbing a suspension of E. coli (ATCC 11775) adjusted to 0.5 McF, 2 antibiotic discs are placed on the surface of the culture medium so that they are centered on the diameter of the agar.

[0158] The antibiotics tested were gentamicin 30 pg (GME 30) (Bio-Rad, 3567318) and a mixture of trimethoprim and sulfamethoxazole (SXT 25) (Bio-Rad, 3568898).

[0159] After incubation for 4 h at 37°C, the cover of the culture medium is removed to measure the diameters of the inhibition zones by analyzing the mCVs emitted on the surface of the agar.

[0160] A NOA electronic nose is attached to the moving part of a robotic axis. A PTFE tube (1 mm internal diameter), connected to the sensor at one end and attached at the other end to a support secured to the moving part of the axis, is positioned at the end of the diameter of the culture medium to be scanned, at a distance of 2 mm above the surface.

[0161] The NOA pump located upstream of the sensor is programmed to have a constant suction flow rate of 30mL / min.

[0162] Signal acquisition is started at the same time as the automated movement of the moving part of the axis. The movement is programmed so that the suction tube travels the diameter of the culture medium from left to right, at a speed of 6 mm / sec (Figure 4).

[0163] The evolution of the average intensity of the signals measured by the NOA sensor when moving the suction tube above the culture medium is presented in the form of a graph (Figure 5). On this graph, arrows indicate the positions of the sensor tubing above the culture medium for which a change in slope translates a change in emission in volatile compounds. Thus, at 7 mm from the starting point, the average intensity decreases due to a disappearance of the detected mCVs, thus marking the beginning of the inhibition zone of the metabolic activity of the E. coli strain for the SXT 25 antibiotic mixture. The arrow positioned at 33 mm marks the position of the tubing from which the average intensity increases, thus reflecting the detection of mCVs and therefore the end of the inhibition zone. Thus, after 4 h of incubation, the observed diameter of the inhibition zone for the SXT 25 antibiotic mixture is 26 mm.

[0164] When the sensor tubing has traveled 47 mm, a further decrease in the average intensity marks the position of a new zone of inhibition of the metabolism of the E. coli strain, ending at a distance of 65 mm from the starting point. After 4 h of incubation, the observed diameter for the inhibition zone for gentamicin is therefore 18 mm.

[0165] Comparison of these inhibition diameter values ​​with the reference values ​​published by EUCAST (RAST 4 hours) allows us to conclude that the E. coli ATCC 11775 strain is sensitive to the antibiotics tested.

[0166] In conclusion, these tests show the possibility of determining the antibiotic susceptibility of a microorganism by implementing an antibiogram by diffusion in agar medium, based on the detection of mCVs, without resorting to a headspace, before growth is observable (Figure 4) and in an automated manner.

[0167] Example 4: Measurement of inhibition diameters for the evaluation of the antibiotic susceptibility of a strain of S. aureus, by local detection of volatile amino compounds emitted from the surface of an agar culture medium.

[0168] The antibiotic susceptibility of the S. aureus strain (ATCC 25923) was assessed using the agar diffusion method. The measurement of the diameters of the inhibition zones, via the analysis of the volatile amino compounds emitted from the surface of the culture medium, was carried out using the device described in Figure 6.

[0169] After inoculation by swab, of a volume of 100 pL of a suspension of S. aureus (ATCC 25923) adjusted to 1 McF, on Mueller-Hinton E agar (BioMérieux, 413822), 4 antibiotic discs are placed on the surface of the culture medium, at a distance of 15 mm from the edge of the Petri dish.

[0170] The antibiotics tested were rifampicin 5 μg (RD5) (Thermo-Scientific, CT0207B), gentamicin 30 μg (CN30) (Thermo-Scientific, CT0072B), a mixture of trimethoprim and sulfamethoxazole (SXT25) (Thermo-Scientific, CT0052B), and a mixture of amoxicillin and clavulanic acid (AMC3) (Thermo-Scientific, CT00538B).

[0171] After incubation for 6 h at 35°C, the culture medium cover is removed and the culture medium is positioned on the rotating tray of the measuring device.

[0172] The open end of the suction tube, connected by its other end to the measuring chamber of the volatile compound sensor, is positioned 1 to 2 mm vertically above an antibiotic disc, via the height adjustment screw of the boom arm.

[0173] The operation of the stepper motor causes the continuous rotation of the culture medium, thus allowing the different zones of the culture medium to be presented under the free end of the suction tube, along a curved linear trajectory passing through the center of each of the 4 antibiotic discs.

[0174] A pump (60 mL / min) located in the NeOse Advance electronic nose housing (Aryba Ile, NOA) and connected to the measuring chamber by a fluidic connection, sucks the locally emitted volatile amino compounds from the surface of the culture medium into the measuring chamber via the open end of the suction tube positioned above the surface to be scanned. Local and continuous detection of the emission of volatile amino compounds from all areas presented under the open end of the suction tube is enabled by the NOA volatile compounds sensor.

[0175] The computer data processing system of the measuring device collects and records: the variations in signal intensity, measured by the sensors sensitive to volatile amino compounds of the NOA volatile compound sensor. These variations reflect differences in volatile amino compound emissions for the different zones of the culture medium thus presented below the free end of the suction tube. The position data (°) of the analyzed zone, delivered by the stepper motor and relative to the starting point.

[0176] Thus, the collected data make it possible to define the local emission profile of volatile amino compounds for the different scanned areas located on the circular trajectory and passing through the center of the 4 antibiotic discs.

[0177] The graphical representation of the intensity (rad) of the measured signal as a function of the position (°) of the analyzed area is given in Figure 7.

[0178] The signal variations, obtained between the 734° and 1120° positions, show variations in the measured signal. An increase in the signal is observed when the area presented under the suction tube approaches an antibiotic disc, then a decrease in this signal is observed when the area presented under the suction tube moves away from the antibiotic disc.

[0179] In this configuration, the inhibition of microbial metabolic activity is translated by an increase in the signal of volatile amino compounds, spontaneously emitted by the culture medium. Thus, for the SXT25 disc, an increase in the measured signal is observed at position 781°, indicating the beginning of the inhibition zone of the S. aureus strain for this antibiotic. A decrease in the signal is observed at position 834°, thus indicating the end of the inhibition zone for the SXT25 disc. The data of the start and end position of the inhibition zone allow to calculate the diameter (D) of the inhibition zone according to the following formula: with AC representing the distance in millimeters (mm), between the center of the culture medium and the center of the antibiotic disc and the term a equal to the difference between the positions in degrees (°) of the end and start of the inhibition zone considered.

[0180] For each antibiotic tested, the start and end positions of the inhibition zones as well as the diameters of the corresponding zones are given in Table 2.

[0181] Table 2: Start and end positions of the inhibition zones for the 4 antibiotic discs tested as well as the diameter values ​​corresponding to each of these zones.

[0182] In conclusion, these tests demonstrate the possibility of determining the antibiotic susceptibility of a microorganism according to the agar diffusion method, using a mapping device for detecting volatile compounds emitted from the surface of the culture medium, without the use of a headspace and before the growth inhibition zones, defined by clearly visible and confluent growth of the S. aureus ATCC 25923 strain, are observable.

[0183] Bibliographic reference

[0184] [1] Patent EP 2376914 B1 in the name of Bio Mérieux Inc. Published on April 19, 2017.

[0185] [2] Perlemoine et al, 2021, “Phage susceptibility testing and infectious titer determination through wide-field lensless monitoring of phage plaque growth”, Pios One, vol. 16, e0248917.

[0186] [3] US Patent Application 2008 / 0199904 A1 in the name of Suslick et al published on August 21, 2008.

[0187] [4] International application WO 2013 / 163610 A1 in the name of Specific Technologies LLC published on October 31, 2013.

[0188] [5] Lim et al, 2014, “Colorimetric sensor array allows rapid detection and simultaneous identification of sepsis-causing bacteria in spiked blood culture”, J. Clin. Microbiol., vol. 52, pages 592-598. [6] Green et al, 2011, “Using a metal oxide sensor (MOS)-based electronic nose for discrimination of bacteria based on individual colonies in suspension”, Sensors and Actuators B: Chemical, vol. 152, pages 21-28.

[0189] [7] Reidt et al, 2020, « Detection of microorganisms with an electronic nose for application under microgravity conditions », Gravitational and Space Research, vol. 8, pages 1-17.

[0190] [8] Maho et al, 2021, « Real-time gas recognition and gas unmixing in robot applications », Sensors and Actuators B: Chemical, vol. 330.

Claims

CLAIMS 1. A method for determining the antimicrobial activity of a product, said method implementing a device comprising a tube (6) comprising a first open end (61) and a second end (62) fluidly connected to a measuring chamber (4); a measuring chamber (4) comprising a gas-sensitive system designed to provide a signal representative of the presence of volatile compounds in a gaseous fluid; and suction means (5) capable of sucking a gaseous fluid present at the first open end of the tube and bringing it from the first open end of the tube into the measuring chamber via the tube; said method comprising the following steps: - seed a surface with microorganisms, - contacting the surface with a solution or a solid support comprising at least one product exhibiting antimicrobial activity, - continuously scanning the surface placed in the open air, before the microbial culture is visible to the naked eye, according to the following sub-steps: a) positioning the first open end of the tube substantially vertically to said surface and at a distance less than or equal to 5R from this surface with R representing the desired spatial resolution, said tube having an internal diameter less than 2R, b) bringing the gaseous fluid present at the surface into the measuring chamber via said suction means, whereby the system sensitive to the gas present in the measuring chamber provides signals characterizing the volatile compounds present in the gaseous fluid brought into the measuring chamber and, from the signals provided, the area inhibition of microorganisms by said at least one product exhibiting antimicrobial activity.

2. Method according to claim 1, characterized in that said surface is an agar culture medium (1) 3. Method according to claim 1 or 2, characterized in that said microorganisms are in the form of a colony, a micro-colony, or a set of isolated microbial cells, visible or not to the naked eye.

4. Method according to any one of claims 1 to 3, characterized in that, when it is the volatile compounds spontaneously emitted by the surface and the emission of which is modulated by microbial metabolic activity which are present in the gaseous fluid brought into the measuring chamber, we observe - a decrease in the signals provided by the gas-sensitive system of the measuring chamber when the first open end of the device tube passes from an inhibition zone to a microbial growth zone, or - an increase in the signals provided by the gas-sensitive system of the measuring chamber when the first open end of the device tube passes from a microbial growth zone to an inhibition zone.

5. Method according to any one of claims 1 to 3, characterized in that, when it is the volatile compounds emitted by microbial metabolic activity which are present in the gaseous fluid brought into the measuring chamber, we observe - an increase in the signals provided by the gas-sensitive system of the measuring chamber when the first open end of the device tube passes from an inhibition zone to a microbial growth zone, or - a decrease in the signals provided by the gas-sensitive system of the measuring chamber when the first open end of the device tube passes from a microbial growth zone to an inhibition zone.

6. Method according to any one of claims 1 to 5, characterized in that the linear speed of the gaseous fluid in said tube is between 0.01 m / s and 10 m / s and advantageously between 0.1 m / s and 5 m / s.

7. Method according to any one of claims 1 to 6, characterized in that said gas-sensitive system comprises at least one gas sensor.

8. Method according to any one of claims 1 to 7, characterized in that said gas sensitive system comprises a plurality of gas sensors.

9. Method according to claim 7 or 8, characterized in that said gas sensor(s) is / are chosen from the group consisting of spectrophotometric gas sensors (Infrared (IR) or UltraViolet (UV)), photoionizing gas sensors (PID), spectrometric gas sensors such as spectrometric gas sensors using selected ion flow tube mass spectrometry (or SIFT-MS for "Selected Ion Flow Tube-Mass Spectrometry") or proton transfer reaction mass spectrometry (or PTR-MS for "Proton-Transfer-Reaction Mass Spectrometry"), electrochemical gas sensors, metal oxide semiconductor gas sensors and sensors based on molecular affinity recognition, whether the transduction mode is optical or gravimetric.

10. Method according to claim 7 or 8, characterized in that said gas sensor(s) is / are one or more optical sensors such as interferometric sensors or plasmonic sensors.

11. Method according to any one of claims 1 to 10, characterized in that said gas-sensitive system further comprises at least one humidity sensor.

12. Method according to any one of claims 1 to 11, characterized in that the scanning of the surface is automated.

13. Device for implementing a method as defined in any one of claims 1 to 12, comprising: a tube (6) comprising a first open end (61) and a second end (62) fluidically connected to a measuring chamber (4); a measuring chamber (4) comprising a gas-sensitive system designed to provide a signal representative of the presence of volatile compounds in a gaseous fluid, said gas-sensitive system being as defined in any one of claims 7 to 10, said gas-sensitive system possibly comprising at least one humidity sensor; suction means (5) capable of sucking a gaseous fluid present at the first open end of the tube and bringing it from the first open end of the tube into the measuring chamber via the tube; and means (3, 7) capable of moving said first open end of the tube and / or a surface to be analyzed.

14. Device according to claim 13, characterized in that it further comprises means capable of locating it in space and / or of locating in space the first open end of the tube.

15. Use of a method as defined in any one of claims 1 to 12 or of a device as defined in claim 13 or 14 for studying the antibiotic susceptibility of microorganisms.

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