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    • 专利摘要:
    A method of detecting a presence or absence of at least a first inhibition zone, said method comprising a step of depositing a volume of the sample in liquid form in a deposition zone extending along an axis on the surface of the agar culture medium and a step of depositing a determined quantity of a chemical agent on the surface of the agar culture medium, said deposit defining a potential zone of inhibition, the axis of the sample deposition area intersecting the potential zone of inhibition.
    公开号:FR3038620A1
    申请号:FR1556490
    申请日:2015-07-09
    公开日:2017-01-13
    发明作者:Laurent Drazek；Agnes Dupont-Fillard；Frederic Pinston；Herve Rostaing
    申请人:Biomerieux SA；
    IPC主号:
    专利说明:

    METHOD FOR DETECTING A PRESENCE OR ABSENCE OF AT LEAST ONE FIRST INHIBITION AREA
    The technical field of the present invention is that of microbiology. More particularly, the present invention relates to the detection of a presence or absence of at least a first zone of inhibition of a sample containing or likely to contain microorganisms in the presence of a chemical agent.
    More particularly, the present invention relates to tests for determining the sensitivity of a microorganism to an antibiotic.
    [0003] A typical sensitivity test is the disk diffusion test, often referred to as the Kirby-Bauer method. This standardized method involves the inoculation of an agar culture medium (for example a 90mm or 150mm Mueller-Hinton agar) with a generally standardized 0.5 Mc Farland sample, obtained from a microbial isolate. The seeding can be performed by conventional manual methods using a swab or oese. Alternatively, seeding can be accomplished by flooding the agar with a standardized suspension to one-tenth of 0.5 Mc Farland, followed by removal of excess sample. Following seeding, one or more paper disks impregnated with defined concentrations of antibiotics are placed on the surface of the agar. After an incubation period, generally 16 to 20 hours at 35 ° C, the diameter of the zone or zones of inhibition around the disks makes it possible to determine the sensitivity of the microorganism present in the seeded sample to each of the agents. antimicrobials impregnated into each disc. Due to the standardization of the Kirby-Bauer method, the results of this method are analyzed by comparing the diameter of each zone of inhibition with the recommendations published by regulatory bodies such as the National Committee for Clinical Laboratory Standards (NCCLS). ) or EUCAST (European Committee on Antimicrobial Susceptibility Testing). The results are thus commonly classified according to one of the following three statements: sensitive, intermediate or resistant. These recommendations therefore result in associated susceptibility reference thresholds corresponding to inhibition zone sizes for each microorganism vis-à-vis each antibiotic.
    By "sensitive" is meant that growth, or survival, microorganisms present in the sample in the presence of the antibiotic is impossible, from a certain concentration of antibiotic. By "intermediate" it is meant that a growth of the microorganisms in the presence of the antibiotic is compromised, starting from a certain concentration of antibiotic. By "resistant" is meant that growth of microorganisms in the presence of the antibiotic is possible, at least up to the threshold of toxicity of the antibiotic for the patient to be treated.
    Another method of detecting sensitivity to antibiotics uses an antibiotic gradient deposited on an agar medium. For this, strips of paper or plastic are impregnated with a concentration gradient antibiotic. The so-impregnated strips are graduated to indicate the antibiotic concentration values present along the strip. One or more strips may be placed on a Mueller-Hinton agar previously seeded in the same manner as the previous method. After incubation, an ovoid area of inhibition of microbial growth around each strip appears if the microorganism present in the sample is sensitive to the antibiotic included in the strip. It is thus possible to deduce a minimum inhibitory concentration (MIC) of microbial growth. The minimum concentration of the antimicrobial agent which makes it possible to inhibit the growth of the microorganism generally retained is thus the readable concentration value on the graduation directly below the point of contact between the zone of inhibition and the long edge of the strip. In other words, the MIC is the visible concentration at the boundary of the zone of inhibition, at the boundary between the microorganism growth zone and the non-growth zone. More particularly, the MIC is legible by identifying the point where the ovoid area of the inhibition zone intersects the strip and locating the corresponding graduation.
    The disadvantage of these methods is that they are difficult to automate. In particular, the flood seeding step requires circular and rippling of the box by the operator to properly distribute the sample deposit over the entire surface of the agar. This particular movement requires a certain technicality of the operator who realizes it and is particularly difficult to reproduce by an automaton. Thus, to be implemented in an automated manner, this method requires visual control means of the deposit to ensure that the entire surface of the agar is covered by the sample. The automation of the deposit is also made complex by the variations in viscosity between the different types of liquid samples that can be used, especially between samples directly from blood cultures and resuspended samples. In addition, the excess sample removal operation also requires means for accurately pipetting and detecting the agar surface. Finally, this method requires large sample volumes, of the order of one milliliter, which increases the biological risk associated with the handling of these samples by the operator.
    The automation of the deposit is also long and tedious with a swab or oese, especially to cover the entire surface of the culture medium.
    On the other hand, automated reading of the inhibition zones is particularly difficult from traditional seeding methods, whether for the method of disks or strips. In particular, the inhibition zones have edges with little pronounced contrast and which can be difficult to locate by an imaging system.
    Finally, another disadvantage is that these methods require a large sample volume and containing a large biomass, including a suspension calibrated at 0.5 Mc Farland 1 mL. This quantity often requires a preliminary step of pre-incubation of the sample in the presence of a broth or an agar medium in order to collect the amount of microorganism colonies required. This preliminary step thus delays the moment when a choice of antibiotic treatment adapted to the type of microorganism present may be performed by the practitioner. It is therefore common for broad-spectrum antibiotics to be delivered while waiting for a sensitivity result, this choice sometimes proving to be ineffective and being known to promote the appearance of resistant microorganisms.
    An object of the present invention is therefore to provide a method for detecting the presence or absence of at least a first inhibition zone making it possible to use a reduced quantity of biomass to be inoculated with respect to traditional methods and, therefore, to reduce the pre-incubation time required for the production of this biomass. More particularly, it is desirable to be able to reliably and quickly characterize a response of microorganisms present or likely to be present in the sample in the presence of the chemical agent. This response is preferably obtained from a culture less than 6 hours and resuspended in a small volume of buffer.
    A second objective of the present invention is to provide an easily automatable method, in particular comprising sample deposition steps and read fast, reliable and repeatable inhibition zones.
    For this, the present invention relates to a method for detecting a presence or absence of at least a first inhibition zone, said method comprising the steps of: a. Provide an agar culture medium; b. Provide a sample containing or likely to contain microorganisms in liquid form; vs. Depositing a volume of the sample in liquid form in a deposit zone extending along an axis on the surface of the agar culture medium; d. Depositing a determined quantity of a chemical agent on the surface of the agar culture medium, said deposit defining a potential inhibition zone, the axis of the sample deposition zone intersecting the potential zone of inhibition; e. Incubate said agar culture medium; f. Determining the presence or absence of said first inhibition zone; The size of the potential zone of inhibition can be defined as the surface of the agar, for example if the sensitivity of the microorganism to the chemical agent is unknown and / or if the microorganism is unknown and / or if the presence of a microorganism in the sample is unknown.
    In other cases where the type of microorganism, for example the genus, species or subspecies, is known, this information provides information on its presumed sensitivity to the chemical agent from recommendations from regulatory bodies such as the National Committee for Clinical Laboratory Standards (NCCLS) or the European Committee on Antimicrobial Susceptibility Testing (EUCAST). These recommendations present for each pair formed of a chemical agent and a given microorganism the size of the potential zone of inhibition obtained after incubation for a given time depending on whether the microorganism is sensitive, intermediate or resistant to the chemical agent.
    Thus, according to an advantageous detection method according to the invention, the sample contains a culture of microorganisms of known type, the area of the potential zone of inhibition then being defined by said type of microorganisms.
    The steps of depositing the sample may especially be automated using a robotic arm or a robotic pipettor having a tool holder moving in translation in three degrees of freedom such as a Hamilton® pipettor Microlab Star. The steps for determining the presence of the zones of inhibition may in particular be carried out using a detection device comprising a light source and a capturing means so as to capture an image of the sample deposited on the medium of culture then by performing a visual examination or automated processing of the image thus obtained.
    The advantage of the invention is therefore to be able to provide an easily automatable method, especially since the step of depositing a volume of the sample in liquid form is carried out according to a deposition zone extending according to a axis at the surface of the agar culture medium. It is thus easy for a programmable automaton to implement this deposition step along an axis whose coordinates are preprogrammed or are determined by conventional imaging methods. On the other hand, the reading of the result of the detection method is also greatly facilitated because the sample, and therefore the zone of inhibition, are located in a smaller area of the culture medium than in a conventional flooding process. or by seeding at the oese. The axis of deposit of the sample will thus be rectilinear or even generally rectilinear. Variations in the deposition axis using curves, portions of curves or consecutive straight portions may be considered if the sample is deposited in the immediate vicinity of the deposition zone of the chemical agent. In particular, in the case of a support having a chemical agent concentration gradient, such as a strip, it may be important to deposit the sample along a generally rectilinear axis, preferably adjacent to the support of the chemical agent. and whatever the form of the support.
    According to a first embodiment, step c) of the detection method according to the invention consists in: c. Deposit a sample volume in liquid form in a continuous line according to a deposit zone extending along an axis on the surface of the agar culture medium.
    The sample volume deposited as a continuous line may have a concentration between 0.0005 Mc Farland and 0.5 Mc Farland. The invention can therefore be applicable to conventional sample concentrations: between 0.5 Mc Farland and 0.1 Mc Farland, low: between 0.1 Mc Farland and 0.01 Mc Farland, or very low: between 0, 01 Mc Farland and 0.0005 Mc Farland. These concentrations can be obtained with a minimum incubation time or without incubation.
    According to a second embodiment, step c) of the detection method according to the invention consists of: c. Deposit a volume of the sample in liquid form into droplets according to a deposit zone extending along an axis on the surface of the agar culture medium.
    A deposit of the sample in discrete form can thus be achieved if the droplets are spaced a distance greater than their diameter once deposited. Advantageously, the droplets deposited are spaced apart by a predetermined pitch, preferably predetermined by making the potential zone of inhibition and / or the diameter of the droplets deposited. For example, the centers of the droplets deposited may be spaced a millimeter, preferably one millimeter in order to be able to quickly compare the number of inhibited drops with the recommendations of regulators. Indeed, these recommendations publish, for a microorganism and a given chemical agent, the size in millimeters of the zone of inhibition measured according to whether the microorganism is sensitive, intermediate or resistant. By counting the number of inhibited drops and / or the number of drops having growth, a sensitivity result can easily be obtained. Thus, in an alternative implementation of the method, it comprises a step of determining the number of inhibited droplets in the potential zone of inhibition and / or the number of uninhibited droplets in the deposition zone of the sample to detect the presence of a potential zone of inhibition and to deduce the sensitivity of the microorganism present in the sample to the chemical agent.
    The volume of each droplet deposited is advantageously between InL and 10pL. The method is therefore applicable to samples of low to very low volumes, including samples from pediatric hospital services. The method also has the advantage of not being too much consumer in sample volume which makes it possible to perform other analyzes from the same sample.
    Thus, the inventors have estimated that an advantageous mode of the process according to the invention could be implemented from a quantity of microorganisms contained in each drop deposited between 1 microorganism per drop and 106 microorganisms per drop, preferably 104 microorganisms per drop. These concentration orders therefore make it possible to analyze samples without incubation or with a limited duration of incubation to reach the necessary concentration.
    The deposition of the chemical agent is for example reduced to a water droplet containing the chemical agent. The detection method according to the invention is thus directly applicable to tests using media impregnated with a chemical agent. Thus, step d) may advantageously consist of: d. Depositing a support impregnated with a determined quantity of a chemical agent on the surface of the agar culture medium, said support defining a potential zone of inhibition, the deposit zone of the sample intersecting the potential zone of inhibition; The deposition of the chemical agent is for example carried out on an impregnated paper or plastic medium. This support may for example be a disc impregnated with the chemical agent, the disc being generally shaped in a cylindrical portion of small thickness. The disc has a quantity of chemical agent which is generally homogeneous in its volume.
    The impregnated support can thus be a disk containing a determined amount of chemical agent. In the case of a disk, the detection method according to the invention may comprise an additional step of measuring the distance between the center of the disk and the first zone of inhibition in order to estimate the sensitivity of the microorganisms contained in the disk. sample to the chemical agent. Advantageously, the axis of deposit of the sample intersects the center of the disc deposited on the medium. Preferably, the process may be continued by a step of classifying the microorganism according to a criteria classification, for example: Sensitive, Intermediate or Resistant, from a sensitivity chart corresponding to the microorganism present in the sample and to the chemical agent. This abacus can in particular be obtained experimentally by learning or from recommendations of regulatory bodies.
    According to a particular embodiment of the invention, step d) consists in: d) performing at least two deposits of a determined quantity of a chemical agent on the surface of the agar culture medium, said deposits each defining a potential inhibition zone, the axis of the sample deposition zone intersecting all the potential zones of inhibition; This particular mode allows in particular to study the effects of synergy between several chemical agents, especially impregnated on disks. For example, two disks comprising two different agents can be deposited on a culture medium, the deposition axis of the sample intersecting the center of these two disks. In another example, four disks comprising four different agents are deposited on a culture medium, the deposition axis of the sample intersecting the center of these four disks so as to follow the edges of a rectangle.
    The impregnated support is for example still an elongated strip of generally rectangular conformation. The strip comprises for example a concentration of chemical agent which follows a gradient of increasing concentration of a small edge to a small opposite edge of the strip. Thus, in an alternative implementation of the detection method according to the invention, the impregnated support is a strip containing a concentration gradient of chemical agent, the deposition of the volume of the sample in liquid form being carried out in parallel and preferably of adjacent to at least one large edge of said strip. Adjacent means that the deposit is made closer to the large edge of the strip without the liquid sample being in contact with the strip. Indeed, it is undesirable that the sample is in direct contact with the strip, this may wet the strip and thus locally modify the diffusion of the chemical in and on the agar.
    Advantageously, the method comprises a further step of: locating a boundary between the first zone of inhibition and the growth zone of the microorganisms determine a minimum inhibitory concentration of the chemical agent from the location of said boundary [ The step of locating a boundary between the first inhibition zone and the growth zone of the microorganisms can be carried out visually or by capturing an image of the culture medium following the incubation step by a acquisition method, then looking for a line or an arc present at the intersection between the growth zone and the zone of inhibition of the sample. According to techniques known to those skilled in the art, the boundary between the inhibition zone and the growth zone can be obtained from an image or a combination of images. This or these images advantageously allowing to display both the graduations present on the strip and the boundary between the zone of inhibition and growth. A conventional technique is to obtain an image in top view of the graduations of the strip and to combine it with a transmission image of the culture medium.
    A conventional method consists, from a digital image, in the definition of a Cartesian coordinate system, typically whose abscissa axis is defined as the main axis of the strip. It is then possible to locate a defined mark of the strip, typically a known text such as a graduation. For example, the characters "256" corresponding to the concentration at 256 μg.ml -1 of chemical agent can be located in the reference. The defined mark is likely to consist of some other and somewhat different indication. The defined mark may also correspond to one of the small edges of the strip. The defined mark then has Cartesian coordinates in the Cartesian coordinate system. In the same way, the large edges of the strip can easily be recognized by conventional image processing means in order to obtain their coordinates in the marker.
    Subsequently, the image is processed to find the boundary between the zone of inhibition and growth. This step may optionally include a smoothing operation to homogenize the image. Such a smoothing operation is for example carried out from a Gaussian filtering. Preferably, the smoothing operation is performed several times, seven times in particular. This step may optionally comprise an operation of dilating the dynamics of the pixel intensity of the image to form a contrast histogram of the image, the contrast being to be considered between dark pixels and clear pixels of the image. This results in a determination of a useful dynamics of the image. This step may then include an image thresholding operation which comprises for example a detection of a threshold and the determination of a contour from a binarization of the image. From this contour, it can then be extrapolated a line or an arc representing the boundary between the growth and inhibition zone, this line or this arc being sought in a pixel zone close to the large edge of the strip, by example less than one centimeter from the big edge.
    From this boundary, an estimation operation of a minimum concentration of inhibition can be carried out. One possible method is to determine the coordinates on the abscissa of the point of intersection between the straight line or the arc obtained and the large edge of the strip. Alternatively, a method may consist in determining the coordinates on the abscissa of the point of intersection between the obtained line or arc and the abscissa axis of the Cartesian coordinate system. Once the abscissa obtained, in relation to the origin of the marker and the known length of the strip, the minimum concentration of inhibition can be determined. In some cases, it is possible for the culture medium to comprise only totally inhibited or totally growing droplets, that is to say where no boundary between a growth and inhibition zone is visible or determinable. In this case, the CMI value can be obtained directly by counting the droplets in relation to the deposit pitch, or from the location of the droplets in the reference mark, in particular the location of the first non-inhibited droplet according to the increasing gradient. concentration of chemical agent. Alternatively, the MIC value can be directly obtained from the location of the last inhibited droplet according to the increasing gradient of chemical agent concentration.
    By culture medium is meant an agar medium, having a layer of agar or the like. The culture media are commonly found in a petri dish or in dehydrated form applied to a support, usually a film. In a nonlimiting manner, other types of culture medium may be used such as media on fibrous media or media on paper.
    The chemical agent is in particular an antibiotic, an antifungal agent, an antimycobacterial agent or a similar compound.
    By likely to contain is meant that the presence of microorganisms in the sample may be suspected from the type of sampling or the symptoms of the patient or the animal on which the sample is taken. However, the type of microorganism likely to be contained in the sample is not known. In the case of mastitis research in cows for example, it may be more effective to directly determine a minimum inhibitory concentration by conventional chemicals before knowing the identification of the type of microorganism present in the sample taken directly from the udder of the cow. Effective treatment of infection with the microorganism can then be prescribed.
    Other features and advantages of the present invention will appear on reading the description which will be made of embodiments, in connection with the figures of the attached plates, in which:
    Figure 1 is a sectional view of a petri dish used for carrying out a detection method of the present invention. FIG. 2 is a schematic view of the detection method of the present invention. Figure 3 is a schematic illustration of sequences of the detection method of the present invention. FIGS. 4a to 4f illustrate a first embodiment of the detection method according to the present invention. FIG. 5 is a schematic illustration of an ombroscopic image pickup device.
    Figures 6a and 6b illustrate images taken in top view of a petri dish used for carrying out an embodiment of the detection method according to the present invention in comparison with a conventional technique.
    Figures 7a to 7f illustrate a second embodiment of the detection method according to the present invention.
    FIG. 8a schematically illustrates a second example of implementation of the invention.
    Figures 8b, 8c and 8d illustrate the second example of implementation of the invention.
    FIGS. 9a to 9d illustrate the second example of implementation of the invention for four different antibiotics - FIGS. 10a, 10b illustrate part of the strips and droplets close to them after 5 hours of incubation of a sample Escherichia coli ATCC 35218 at 0.5 Mc Farland and 0.01 Mc Farland in the presence of ampicillin / sulbactam. FIGS. 11a and 11b illustrate a third method of implementing the invention
    FIG. 12 illustrates a visual examination after 6:30 of incubation of a medium inoculated according to the third method of implementation of the invention
    FIG. 13 illustrates an example of a sampling tool that can be used according to the third method of implementation of the invention. Referring to FIGS. 1 and 3, in the medical and / or pharmaceutical field, it is frequently having to use a method for detecting the presence or absence of an inhibition zone on a culture medium 2 of a sample 1 in the presence of a chemical agent soaked on a support 3. The culture medium 2 is contained in a petri dish 4 and receives the sample 1 containing or likely to contain the microorganisms, as well as the chemical agent 5 which is capable of inhibiting a development of certain microorganisms. The microorganisms are chosen indifferently from bacteria, yeasts or fungi. Alternatively, the method according to the present invention can be applied to plant or animal cells. The culture medium 2 is preferably an agar, a layer of agar or the like. The culture medium 2 may also be a dehydrated culture medium on paper or fibrous support. The medium is then rehydrated by the sample. The chemical agent 5 is especially an antibiotic, antifungal, an antimycobacterial or a similar compound.
    More particularly, it is desirable to be able to reliably and quickly characterize a response of the sample containing or likely to contain the microorganisms 1 to the presence of the chemical agent 5, such a response being commonly classified according to the one of three statements: sensitive, intermediate or resistant. It may also be desirable to obtain a minimum inhibitory chemical concentration value that can inhibit the growth of microorganisms present in the sample.
    Such a detection method 100 finds particularly frequent applications in the field of medical, pharmaceutical and / or veterinary diagnostics implemented for the detection of a pathology in a patient or an animal. As a result, such a detection method 100 is reliably desired in the sense that the nature of the aforementioned response of the microorganisms contained or likely to be contained in the sample 1 to the chemical agent 5 is certain, undoubtedly and without ambiguity. It also follows that such a detection method 100, whose successive sequences are illustrated in FIGS. 2 and 3, is desired fast with a response time, which flows between an initial time T0 at which the sample is set. in contact with the chemical agent 5 and a TX detection time at which said reliable response is obtained, which is desired as short as possible, and is especially less than eight hours. It also follows that it is desirable for such a detection method 100 to have adequate repeatability. Such objects are advantageously achieved from the implementation of the detection method 100 of the present invention.
    In general, and with reference to FIG. 3, the detection method 100 of the present invention comprises a step a) of supplying an agaric culture medium 2, a step b) of supplying a Sample 1 containing or likely to contain microorganisms in liquid form. For example a raw sample directly taken from the patient. In this case, the raw sample may undergo a preculture phase allowing isolation of strains of microorganisms in the presence, for example in the case of a sample comprising a diversity of microorganisms, and incubation of selected and isolated microorganisms. Sample 1 is for example still a prepared sample, in particular filtered, centrifuged and / or purified in a similar manner. Sample 1 may have a biomass of microorganisms that is sufficient to be validly analyzed, such as a standard of concentration between 0.0005 Mc Farland and 0.5 Mc Farland. Sample 1 is for example urine, blood, cerebrospinal fluid or a similar biological fluid.
    A volume of the sample in liquid form is then deposited in step c) according to a deposit zone extending along an axis on the surface of the agar culture medium. The sample may be deposited in droplets, in particular using a manual pipette or a pipetting automaton. The droplets may advantageously have an identical volume and be spaced apart by a predetermined pitch P, where P is the distance between the centers of two consecutive droplets. In the case where the diameter of the droplets once deposited is greater than the value of the pitch P, the droplets then form a liquid deposit in a continuous line.
    The sample may also be deposited by a swab, in particular a flocked or fibrous swab soaked in a volume of the sample and then moved in contact with the agar medium along an axis. Alternatively, a pipetting device comprising filtration means as described in the international application published under the number W02012 / 083150 A2 can be used to pipette and filter a sample volume and deposit it by friction in contact with the surface of the medium. of agar culture. The displacement of the swab or the pipetting device comprising filtering means described above in contact with the agar thus makes it possible to form a deposition of the sample in a continuous line, this line extending along an axis.
    The process is continued in step d) by the deposition of a determined amount of a chemical agent on the surface of the agar culture medium, said deposit defining a potential zone of inhibition, the axis of the sample deposition area intersecting the potential zone of inhibition. Alternatively, this deposit can be made before the deposit of the sample. The initial moment T0 is considered as the moment when the sample and the chemical agent are brought into contact on the surface of the culture medium.
    The process continues in step e) by incubating the agar culture medium.
    The method continues in step f) which consists in determining at a time Tx of incubation the presence or absence of said first zone of inhibition of the sample around the deposition zone of the chemical agent, in the potential zone of inhibition.
    A first example of implementation of the invention will be detailed in Figures 4a to 4f illustrating Petri dishes 4 in top view.
    Referring to Figure 4a, the first embodiment of the detection method according to the present invention comprises a step of providing an agar culture medium 2 in a Petri dish 4.
    The deposition of a disc 3 impregnated with a predetermined quantity of a chemical agent 5 is carried out on the surface of the agar culture medium, said deposit defining a potential circular inhibition zone 6 around the disc.
    According to Figure 4b, a deposition axis 7 intersecting the potential inhibition zone 6 and the center of the disc 3 is defined. This axis also defines a deposition zone 8 of the sample 1 which is generally rectangular and equitably distributed on either side of the deposition axis.
    According to FIG. 4c, a liquid sample 1 is deposited at T0, in the form of multiple droplets, at the surface of the culture medium 2, in the deposition zone 8 and along the axis 7. The droplets are spaced apart from each other. a pitch P, corresponding to the distance between the center of two consecutive droplets. Incubation of the culture medium then begins.
    According to Figure 4d, at time T1 after an incubation time, the presence of a first inhibition zone 9 of the sample 1 is determined, for example by visual analysis. In fact, some droplets show bacterial growth, while others show no bacterial growth. At the intersection between the first inhibition zone 9 and the sample deposition zone 8, certain droplets have a growing portion and an inhibited portion 1b. It is thus possible at this time to demonstrate the presence of microorganisms in the sample as well as the inhibition of the growth of these microorganisms in the presence of the chemical agent 5. The inhibition zone 9 has a diameter D which can notably be measured by a vernier caliper.
    According to Figure 4e, at time T2 after a longer incubation time, the surface of the first inhibition zone 9 and its diameter D are stable and no longer vary. It is thus possible at this time to reliably measure the size of the inhibition zone. This determination is particularly easy in the case of Figure 4e where some droplets have bacterial growth la, while others show no bacterial growth, the simple counting operation, with respect to the pitch P between each drop allows to determine the sensitivity of the microorganism to the chemical agent. On the other hand, by obtaining an identification of the microorganism in the presence and comparing the size of the zone of inhibition with the recommendations of the regulating organisms for the chemical agent 5, microorganism in the presence, the classification of the strain as "sensitive "," Intermediate "or" resistant "is also possible.
    According to Figure 4f, it is likely that at a time T2, the first zone of inhibition 9 is no longer visible or greatly reduced and that some or all of the droplets have a bacterial growth la. It is thus possible at this time to determine that the type of microorganism present in the sample 1 is resistant to the chemical agent 5. It is also possible that no inhibition zone is apparent and this whatever the duration incubation, demonstrating the resistance of the microorganism to the chemical agent.
    In order to implement a method according to the invention, it may for example be used a capture device comprising a capture means and a light source so as to capture an image of the sample deposited on the culture medium. in the presence of a chemical agent.
    An exemplary capture device 24 by ombroscopy is illustrated in Figure 5. This device comprises a pickup means 25. The pickup means 25 comprises a pickup axis A which is preferably arranged orthogonally with respect to a first plane PI according to which is extended the culture medium 2. The capturing means 25 advantageously overhangs the petri dish 4 so as to take a top view of the culture medium 2. The capturing means is for example a CCD camera, particularly of type Basler piA2400 - 17 gm, which is equipped with a telecentric lens 23. The light source 20 is preferably a collimated illuminator capable of producing parallel light rays 21 between them which reach orthogonally the culture medium 2 after being reflected by a mirror 22. The light source 20 may comprise a plurality of diodes having a range of emission indifferently in the red, the v Er, blue and white. The light source 20 is, for example, of the Opto Engineering - LTCL 048 - W type. The telecentric objective 23 is in particular of the Opto Engineering - TC23 048 type, comprising a focal field of 46x38.5 mm and a working distance of 134.6. mm. Advantageously, the device 24 may comprise calculation means 26 comprising, for example, analysis and image processing means, the calculation means 26 constituting a processor that comprises the capture device 24. Advantageously, the device 24 may comprise one or other light source (s) (not shown) arranged (s) above the culture medium and directed (s) to the culture medium. These sources make it possible, for example, to more optimally illuminate the printed part of the support impregnated with chemical agent, in particular so as to locate more easily a defined mark of the support such as one or more characters printed on the support.
    Figures 6a and 6b, compare the results of a traditional method of detecting a presence or absence of at least a first zone of inhibition with the method according to the invention. To carry out this comparison, two Mueller Hinton E agar plates (bioMérieux Ref 413822) are inoculated with an inoculum obtained from a culture of Staphylococcus aureus ATCC 25923 (American Type Culture Collection) at a concentration of 0.5 Mc Farland. The first agar is inoculated by flooding with a volume of approximately 1 ml, while on the surface of the second agar are deposited ten 3pL droplets separated by a pitch of 5 mm along an axis 7 in a deposition zone. Impregnated disk 3 containing 1 ampicillin Opg is also deposited on each of the agar plates. In FIG. 6b, the disk is deposited so as to intersect the droplet deposition axis 7 and the sample deposition zone 8. The two agars are then incubated at 37 ° C for 6 hours 30 minutes. Following this incubation time, an image in top view of each agar is captured using a device 24 for capturing by shadoscopy as previously described and making it possible to obtain the images 27a and 27b of FIGS. 6b. At the capture time of the images 27a and 27b, it is thus possible to observe a diameter D, corresponding to a zone of inhibition, similar between the methods and greater than the reference threshold R defined by the EUCAST. Indeed, the diameter D of the zone of inhibition is 22mm, the reference threshold R between sensitive and resistant defined by EUCAST being equal to 18mm. The method according to the invention therefore makes it possible to use a minimum volume of sample while obtaining an identical result of sensitivity to a chemical agent. Many techniques known to those skilled in the art can be used to determine the diameter D in an automated manner. For example, a method may consist of locating the drop deposition axis and the center of the disc from a digital image of the culture medium. Subsequently, it is possible to extract an intensity profile of the pixels of the image along an axis passing through the center of the disk and along or parallel to the droplet deposition axis. From this intensity profile, we then look for the most important contrast transitions on both sides of the disc. These transitions can in particular be sought by searching for the position in the profile of the first rising edge, or the maximum value of the first derivative of the profd. The two positions obtained on either side of the disk then correspond to the diameter D of the desired inhibition zone.
    A second example of implementation of the invention will be detailed according to Figures 7a to 7f.
    Referring to FIG. 7a, the second example of implementation of the detection method according to the present invention comprises a step of supplying an agar culture medium 2 in a petri dish 4.
    The deposition of a strip 3 impregnated with a predetermined quantity of a chemical agent 5 is carried out on the surface of the agar culture medium, said deposit defining a potential zone of ovoid inhibition 6 around the strip.
    According to Figure 7b, a deposition axis 7 intersecting the potential zone of inhibition 6 and parallel to one of the large edges 3a of the strip 3 is defined. This axis also defines a deposition zone 8 of the generally rectangular sample and equitably distributed on either side of the deposition axis.
    According to FIG. 7c, a liquid sample 1 is deposited at T0, in the form of multiple droplets, at the surface of the culture medium 2, in the deposition zone and along the axis 7. The droplets are spaced apart from each other. a pitch P, corresponding to the distance between the center of two consecutive droplets. Incubation of the culture medium then begins.
    According to Figure 7d, at time T1 after an incubation time, the presence of a first inhibition zone 9 of the sample 1 is determined, for example by visual analysis. In fact, some droplets show bacterial growth, while others show no bacterial growth. At the intersection between the first inhibition zone 9 and the sample deposition zone 8, certain droplets have a growing portion and an inhibited portion 1b. It is thus possible at this time to demonstrate the presence of microorganisms in the sample as well as the inhibition of the growth of these microorganisms in the presence of the chemical agent 5.
    According to Figure 7e, at time T2 after a longer incubation time, the surface of the first inhibition zone 9 is stable and no longer varies. It is thus possible at this time to reliably measure the size of the zone of inhibition and to deduce the value F corresponding to the minimum inhibitory concentration of the chemical agent. This value F corresponds to the concentration of chemical agent soaked on the strip at the intersection between the zone of growth and inhibition of the sample.
    In the case where the medium has only droplets with bacterial growth la, or without bacterial growth, the value F corresponds to the intermediate value obtained by drawing a perpendicular to the axis 7 between the last inhibited droplet and the first droplet growing in the direction of the increasing concentrations of the strip and observing the corresponding chemical agent concentration value on the strip.
    In the case where the medium has a droplet with a growing portion and an inhibited portion lb, the value F may in particular be obtained by looking for an arc or a straight line at the boundary between the growing portion and the inhibited portion; by looking for the intersection of this arc or this straight line with the large edge 3a of the strip and observing the chemical agent concentration value at this intersection. Alternatively, the droplets having a growing portion and an inhibited portion 1b may be ignored to search for the first higher concentration value that completely inhibits the growth of a droplet.
    According to Figure 7f, it is possible that at a time T2 the first inhibition zone 9 is no longer visible or reduced and that some or all of the droplets have a bacterial growth la. It is thus possible at this time to determine that the microorganism present in the sample 2 is resistant to the chemical agent 5. It is also possible that no inhibition zone is apparent and this regardless of the duration of incubation, demonstrating the resistance of the microorganism to the chemical agent.
    Figures 8a to 8d and 9a to 9d illustrate this second example of implementation of the invention. According to FIG. 8a, a sample 1 is deposited in the form of a series of droplets, along an axis parallel to the large edge of a strip 3 containing a chemical agent concentration gradient. The strip 3 comprises graduations of chemical agent concentrations 13 to deduce a minimum inhibitory concentration of chemical agent. An example of such a strip is marketed by the applicant under the trademark Etest®. According to Figure 8b, a strip 3 containing a chemical agent 5 is deposited on a culture medium 2 contained in a Petri dish 4. The detail B of Figure 8b is visible in Figure 8c. A sample 1 is deposited in the form of a series of droplets, in a deposition zone 8 along an axis 7 parallel to the large edge of the strip 3. In this example, drops of 13 nanoliters are deposited by a pipeteur Pipejet P9 Nanodispenser marketed by the company biofluidix and mounted on a robotic arm. Detail C of Figure 8c is visible in Figure 8d. 50 droplets are thus deposited along the axis 7 with a pitch P of 800 μm. Each droplet covers an area of about 1mm2.
    According to a first experiment of this example, an inoculum of a strain of Escherichia coli ATCC 35218 (American Type Culture Collection) having a concentration of 0.5 Mc Larland is thus deposited in droplets along four Etest® strips. (bioMérieux), each deposited on an agar culture medium Mueller Hinton E (bioMérieux). The strips respectively contain a concentration gradient of: gentamicin, tetracycline, ampicillin / sulbactam, ampicillin. Similarly, an inoculum of a strain of Escherichia coli ATCC 25922 with a concentration of 0.5 Mc Farland is deposited in droplets along four Etest® strips (bioMérieux), each deposited on a Mueller agar culture medium. Hinton E (bioMérieux). The strips also contain a concentration gradient of: gentamicin, tetracycline, ampicillin / sulbactam, ampicillin.
    Fifty droplets, a volume of 13 nanoliters each, and spaced 800 microns are deposited along each strip. Depending on the concentration of 0.5 Mc Farland, it is estimated that about 2000 bacteria are present in each droplet. The eight culture media thus prepared are then incubated and monitored periodically to determine the presence or absence of a zone of inhibition and / or growth of microorganisms.
    Figures 9a to 9d illustrate in top view, the four environments where the sample of Escherichia coli ATCC 35218 was deposited. FIGS. 9a to 9d are obtained using a shadoscopic recording device as presented with reference to FIG. 5. FIG. 9a shows a view after 5 hours of incubation of the sample in the presence of gentamicin. The sample then has growing droplets 1a, droplets in which the growth is inhibited, and at least one partially inhibited droplet 1b. From conventional techniques of image analysis of the partially inhibited droplet 1b, a straight line 14 is obtained perpendicular to the large edge of the strip and delimiting the boundary of the growth zone of the microorganisms and the zone of inhibition. . A minimum inhibitory concentration value is then obtained at the intersection between the line 14 and the large edge of the strip. A minimum inhibitory concentration (MIC) of 2 μg / ml is then determined.
    Advantageously, a straight line or an arc delimiting the boundary of the growth zone of the microorganisms and the zone of inhibition may be sought, this line not necessarily being perpendicular to the major edge and thus restoring more faithfully. said boundary. This line or arc corresponds to a portion of the ovoid inhibition area observed with conventional seeding methods and therefore allows a better estimate of the MIC. In the case where a straight line or an arc is sought, the minimum inhibitory concentration value can be obtained by drawing a perpendicular to the large edge of the strip intersecting the point of intersection between the line 15 and the deposition axis of the line 15. sample 7, then looking for the corresponding graduation at this perpendicular on the strip [0074] FIG. 9b shows a view after 5 hours of incubation of the sample in the presence of tetracycline. A similar procedure is repeated and makes it possible to estimate a minimum inhibitory concentration of 1.5 mg / ml. Figure 9c shows a view after 5 hours of incubation of the sample in the presence of ampicillin / sulbactam. A similar procedure is repeated and makes it possible to estimate a minimum inhibitory concentration of 18 mg / ml. Figure 9d shows a view after 5 hours of incubation of the sample in the presence of ampicillin. In this figure, only growing droplets are observed, demonstrating the resistance of the microorganism to ampicillin, the observed value of minimum inhibitory concentration is then greater than 256 μg / ml.
    The results of minimum inhibitory concentrations obtained for Escherichia coli strains ATCC 35218 and strains of Escherichia coli ATCC 25922 are compared with the conventional method by flooding the medium in Table 1 below. The values are given in μg / ml at different incubation times. The inhibition values for the flood method are measured from images obtained by a shadoscopic capture device as described above. The inhibition values for the method according to the invention are measured from images obtained by a shading device at 5 o'clock and visually at 7 o'clock.
    Table 1, R = Resistant, values in gg / ml Table 1 makes it possible to conclude to a good correlation of the MICs estimated between the conventional method and the method according to the invention. It is noted that small differences in the values obtained can be observed in the case where the deposition of the droplets is too far from the large edge of the strip or made non-parallel to the large edge. An optimal positioning at 1mm of the strip of the sample deposition axis and parallel to the large edge thus makes it possible to limit differences in estimated MIC values. On the other hand, it is clearly established that the ejection of the sample by the automated pipettor PipeJet® P9 Nanodispenser (bioFluidix) does not prevent the growth of the bacteria tested, and this experiment also demonstrates that it is possible to to estimate a MIC with a sample of reduced volume, the total number of microorganisms deposited per culture medium being here estimated at 100,000 bacteria for the method according to the invention Finally, a very strong optical contrast is observed at 5 o'clock in the shadow and at 7 hours in visual analysis, to accelerate the method of determination of the minimum inhibitory concentration by depositing the sample in droplets.This very high contrast also makes it possible to consider the use of conventional techniques of analysis of image to automatically determine this concentration value.
    A second experiment of this example is conducted to evaluate the behavior of the method according to the invention with samples of very low concentrations. For this, three suspensions of Escherichia coli ATCC 35218 respectively at 0.5 Mc Farland, 0.1 Mc Farland and 0.01 McFarland are prepared. The MIC values are evaluated for each of these strains in the presence of a strip of gentamicin, tetracycline, ampicillin / sulbactam or ampicillin. For this, fifty droplets of 18 nanoliters each are arranged along an axis parallel to the large edge of each strip, similar to the first experiment and for each of the concentration values. Figure 10a illustrates a portion of the strip and droplets proximate thereto after 5 hours of incubation of the Escherichia coli ATCC 35218 sample at 0.5 Mc Farland in the presence of ampicillin / sulbactam. Figure 10b illustrates a portion of the strip and droplets thereon after 5 hours of incubation of the Escherichia coli ATCC 35218 sample at 0.01 Mc Farland in the presence of ampicillin / sulbactam. Figures 10a and 10b are obtained by a shadoscopic capture device as presented above. These two figures make it possible to determine the presence of a zone of inhibition and to estimate a MIC at 10 mg / ml, even at a very low concentration. Table 2 below presents the results of this second experiment in comparison with a classical flooding method.
    Table 2, R = Resistant, values in pg / ml This second experiment also makes it possible to conclude to a good correlation between the MICs determined by the droplet method according to the invention and the flooding method. This experiment also demonstrates that the method according to the invention makes it possible to estimate a MIC with a sample of reduced volume and low concentration, the total number of microorganisms deposited per culture medium being here estimated at 2000 bacteria, ie 50 drops containing in average 40 bacteria. Finally, it is also observed a very strong optical contrast at 5 hours in shadow and at 7 hours in visual analysis, to accelerate the method of determining the minimum inhibitory concentration.
    A third method of implementation of the invention is illustrated by FIGS. 11a, 11b and 12. In this method, the sample is deposited by friction on the surface of the culture medium with the aid of FIG. a sampling tool as described in the international application published under the number W02012 / 083150. Such an integrated device makes it possible to carry out one or more filtration steps as well as to transfer a liquid sample. Said device comprises a tubular part whose one end is covered with a filtration material such as a membrane, said filtration material being disposed outside this end and covering it completely. The other end of the tubular portion is adapted to be connected with a suction or dispensing means such as a vacuum pump. The advantage of the use of such a tool is in particular to be able to estimate the MIC of a sample from a bottle of positive blood culture, that is to say presenting a result attesting the presence of microorganisms after a time of given incubation. For this purpose, the sample may in particular be prepared by taking a volume from the positive blood culture, by performing a step of lysing the blood cells, for example chemical, of the volume taken, then by proceeding with suction filtration at the surface of the blood sample. a membrane of the lysed volume. The microorganism concentrate can then be deposited directly on a culture medium by affixing the membrane covering the end of the sampling tool on the agar surface of the culture medium and then moving it on the surface of the culture medium 2 according to a axis 7 and as shown in Figure 11a. The operation can be renewed in order to deposit the sample along a second axis 7 '. A strip 3 containing a chemical agent is deposited parallel to the axis 7 and advantageously between the two axes 7 and 7 '. The surface covered by the deposition zone or zones 8, 8 'must then preferably be adjacent to the edge of the strip so as not to be in direct contact with it and to avoid chemical agent diffusion effects.
    An example of this third implementation method of the invention will now be described. An Etest® strip of Gentamicin (bioMérieux) is deposited on a Mueller Hinton E agar 2 (bioMérieux, Ref 413822). A suspension calibrated at 0.5 Mc Farland of Escherichia coli ATCC 25922 is prepared.
    In order to perform this operation in an automated manner, the support of the agar is placed on a motorized stage moving in translation along an axis X, the axis of the strip being parallel to this axis. The sampling tool is supported by a motorized arm moving in translation along a substantially vertical axis Z. In connection with FIG. 13, the sampling tool 30 comprises a body 32 of tubular shape comprising an end covered with a PES Supor® membrane 34 with a porosity of 0.45 μm (Pall). The other end 42 is open to connect a suction means such as a vacuum pump. In the body 32 and between the membrane 34 and the end 42 are arranged: a fibrous portion 36 such as cotton, a set of glass ball 38 whose diameter is between 212pm and 300μηι, the balls being held by a part fibrous 40 cotton. The interest of these different parts is described in more detail in the international patent application published under the number W02012 / 083150. The following protocol is used to prepare the sample: • 1 mL of a positive blood culture bottle is removed and mixed with three suction / dispensing cycles at 0.5 mL of lysis buffer (0.45% w / v). Brij-97 + 0.3M CAPS, pH 11.7) using a pipette in a container • The mixture is incubated for two minutes at room temperature to obtain a lysed sample • Following incubation, the end supporting the membrane 34 of the sampling tool 30 is immersed in the lysed sample and then filtered by suction at -600mbar for 2 minutes • The sampling tool is then moved to another container comprising a first washing solution (Brij / Solution Saline (0.45% w / v NaCl + 0.05% Brij 97)) • The end supporting the membrane 34 of the sampling tool 30 is immersed in the first washing solution and then aspirated (at -600mbar) for 4 minutes • L picking tool It is then moved to another container comprising a second washing solution (deionized water). The end supporting the membrane 34 of the sampling tool 30 is immersed in the second washing solution and sucked (at -600mbar) for 3 minutes. minutes then 20 seconds of aspiration outside the container to limit the appearance of bubbles in the sampling tool 30. • The microorganisms contained in the sample are thus concentrated on the surface of the membrane 34 [0082] In accordance with Figure 1a, the sample 1 thus prepared by filtration is then deposited by displacement of the Z membrane 150pm from the surface of the agar so as to introduce it very slightly into the agar. The membrane is then moved along the X axis at 65mm constant Z. In order to accurately measure the Z-displacement of the sampling tool, conventional techniques for detecting the surface of the agar may be employed.
    The sample is thus deposited along two axes 7, 7 'adjacent and parallel to the large edges of the strip 3 Etest® Gentamicin. Thus, the two deposition zones 8, 8 'obtained have a uniform distribution and adjacent to the large edges of the strip.
    Each deposit zone has a width of about 2mm. The culture medium thus seeded is incubated at 35 ° C. Images in a top view of the medium are captured using a shadow capture device at regular times. A visual examination is also performed. Figure 11a shows an image captured by shadoscopy after 3:30 of incubation. Figure 11b shows an image captured by shadoscopy after 6:30 of incubation. Figure 12 is a photograph of the same box in natural lighting after 6:30 of incubation.
    It is thus possible from 3.30 hours of incubation to detect the presence of an inhibition zone of the sample and a growth zone of the microorganisms present in the sample 1a. It is also possible to determine a minimum inhibitory concentration of 1.5 μg / ml from images obtained by ombroscopy. A visual examination at 6:30 also confirms this value as illustrated in Figure 12. The advantage of this method is therefore to be able to determine a MIC from a very small sample volume and with a limited number of manipulation and easily automatable. The deposition movement on the surface of the agar may also be carried out by a robotic arm or a tool-holder moving in translation along three axes of freedom.
    The methods and devices described in the present invention may be implemented by one or more computer programs, which may be presented in various forms, active or inactive, on a single computer or distributed on computer systems. computer. For example, they may be implemented by software comprising instructions able to implement the methods of the present invention and described in the form of source code, object code, executable code or any format allowing the realization of certain process steps. according to the invention, in particular the steps of: • Determining the presence or absence of said first inhibition zone. • Determine the number of inhibited droplets in the potential inhibition zone and / or the number of uninhibited droplets in the sample deposition area. • Measure the distance between the center of the disc and the first inhibition zone in order to estimate the sensitivity of the microorganisms contained in the sample to the chemical agent. Classify the microorganism according to a three-criteria classification: Sensitive, Intermediate or Resistant, starting from a sensitivity chart corresponding to the microorganism present in the sample and to the chemical agent, this abacus being for example available on a storage medium. Locate a boundary between the first zone of inhibition and the zone of growth of the microorganisms. Determine a minimum inhibitory concentration of the chemical agent starting from the localization of said border. All these computer programs can be stored on a readable storage medium for a computer, which includes the storage media and corresponding signals, in a compressed or uncompressed form.
    The term computer refers to any electronic device comprising a processor, such as a central processing unit (CPU), a dedicated processor or a microcontroller. A computer is capable of receiving data (one or more inputs), performing a sequence of predetermined steps on that data, and producing a result in the form of information or signals (one or more outputs). Depending on the context, the term computer may mean a particular processor or more generally a processor associated with an assembly of interconnected elements contained in a single housing.
    The term readable storage medium for computer or storage medium refers to any means of containing, storing, communicating, distributing, or transporting the computer program for use by or in connection with a computer or any other means of execution of said program. The readable computer storage medium may be, without limitation, an electronic, magnetic, optical, electromagnetic, infrared or semiconductor system and an apparatus, device or means for propagating said program. More specific and non-limiting examples of storage media may be a diskette, a CD-ROM, random access memory (RAM), read-only memory (ROM), programmable integrated read-only memory (EPROM or FLASH storage), a fiber optical or any electrical connection comprising one or more cables.
    [0090] The invention also relates to a system comprising a computer and one or more computer programs configured to implement one or more methods according to the invention. Advantageously, said system also comprises means for controlling a capture device able to capture images of the culture medium after incubation, the captured images being processed by said computer program. Advantageously, said system also comprises means for moving the culture medium such as motorized plates and the control means of these displacement means. Advantageously, said system also comprises automated sample deposition means such as robotic arms, robot pipettors, etc., as well as the control means of these depositing means.
    权利要求:
    Claims (16)
    [1" id="c-fr-0001]
    claims
    A method of detecting a presence or absence of at least a first inhibition zone (9), said method comprising the steps of: a. Provide an agar culture medium (2); b. Provide a sample (1) containing or likely to contain microorganisms in liquid form; vs. Depositing a volume of the sample (1) in liquid form according to a deposition zone (8) extending along an axis (7) on the surface of the agar culture medium; d. Depositing a determined quantity of a chemical agent (5) on the surface of the agar culture medium (2), said deposit defining a potential inhibition zone (6), the axis of the deposition zone of the sample intersecting the potential zone of inhibition; e. Incubate said agar culture medium (2); f. Determining the presence or absence of said first inhibition zone (9);
    [2" id="c-fr-0002]
    2. Detection method according to the preceding claim, the sample containing a culture of microorganisms of known type, making the potential zone of inhibition being defined by said type of microorganisms.
    [3" id="c-fr-0003]
    3. Detection method according to one of the preceding claims, characterized in that step c) consists of: c. Deposit a sample volume in liquid form in a continuous line according to a deposit zone extending along an axis on the surface of the agar culture medium;
    [4" id="c-fr-0004]
    4. Method according to the preceding claim, characterized in that the concentration of the sample is between 0.0005 Mc Farland and 0.5 Mc Farland
    [5" id="c-fr-0005]
    5. Detection method according to one of the preceding claims, characterized in that step c) consists of: c. Deposit a volume of the sample in liquid form into droplets according to a deposition zone extending along an axis on the surface of the culture medium;
    [6" id="c-fr-0006]
    6. Detection method according to the preceding claim, characterized in that the deposited droplets are spaced apart by a predetermined pitch P, preferably predetermined by the area of the potential inhibition zone and / or the diameter of the droplets deposited.
    [7" id="c-fr-0007]
    7. Method according to the preceding claim, characterized in that the droplets deposited are spaced by a millimeter, preferably a millimeter
    [8" id="c-fr-0008]
    8. Detection method according to claims 5 to 7, characterized in that the volume of each droplet deposited is between InL and 10gL
    [9" id="c-fr-0009]
    9. Process according to any one of the preceding claims, characterized in that the quantity of microorganisms contained in each drop deposited is known and between 1 microorganism per drop and 106 microorganisms per drop, preferably 104 microorganisms per drop.
    [10" id="c-fr-0010]
    10. Method according to one of claims 5 to 9, characterized in that it comprises a step of - determining the number of inhibited droplets (Ic) in the potential zone of inhibition and / or the number of droplets not inhibited (la) in the sample deposition zone;
    [11" id="c-fr-0011]
    11. Detection method according to any one of the preceding claims, characterized in that step d) consists of: d. Depositing an impregnated support (3) with a determined quantity of a chemical agent on the surface of the agar culture medium, said support defining a potential inhibition zone, the deposit zone of the sample intersecting the potential zone of inhibition;
    [12" id="c-fr-0012]
    12. Detection method according to the preceding claim, characterized in that the impregnated support is a disc containing a determined amount of chemical agent.
    [13" id="c-fr-0013]
    13. Detection method according to the preceding claim, characterized in that it comprises an additional step of: measuring the distance between the center of the disk and the first zone of inhibition in order to estimate the sensitivity of the microorganisms contained in the sample to the chemical agent;
    [14" id="c-fr-0014]
    14. The detection method according to the preceding claim, characterized in that it comprises a further step of: classifying the microorganism according to a classification with three criteria: Sensitive, Intermediate or Resistant, from a sensitivity chart corresponding to the microorganism present in the sample and the chemical agent;
    [15" id="c-fr-0015]
    15. The detection method as claimed in claim 11, characterized in that the impregnated support is a strip containing a chemical agent concentration gradient, the deposition of the volume of the sample in liquid form being carried out in parallel and adjacent to the large one. edge of said strip;
    [16" id="c-fr-0016]
    16. The detection method according to the preceding claim, characterized in that it comprises a further step of: locating a boundary between the first zone of inhibition and the growth zone of the microorganisms; determining a minimum inhibitory concentration of the chemical agent from the location of said boundary;
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    同族专利:
    公开号 | 公开日
    CN108474019A|2018-08-31|
    EP3320109B1|2020-03-18|
    FR3038620B1|2019-05-24|
    WO2017006055A1|2017-01-12|
    EP3320109A1|2018-05-16|
    US10870876B2|2020-12-22|
    US20180208958A1|2018-07-26|
    CN108474019B|2022-02-01|
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    法律状态:
    2016-07-26| PLFP| Fee payment|Year of fee payment: 2 |
    2017-01-13| PLSC| Publication of the preliminary search report|Effective date: 20170113 |
    2017-07-26| PLFP| Fee payment|Year of fee payment: 3 |
    2018-07-26| PLFP| Fee payment|Year of fee payment: 4 |
    2019-07-25| PLFP| Fee payment|Year of fee payment: 5 |
    2020-07-27| PLFP| Fee payment|Year of fee payment: 6 |
    2021-07-26| PLFP| Fee payment|Year of fee payment: 7 |
    优先权:
    申请号 | 申请日 | 专利标题
    FR1556490A|FR3038620B1|2015-07-09|2015-07-09|METHOD FOR DETECTING A PRESENCE OR ABSENCE OF AT LEAST ONE FIRST INHIBITION AREA|
    FR1556490|2015-07-09|FR1556490A| FR3038620B1|2015-07-09|2015-07-09|METHOD FOR DETECTING A PRESENCE OR ABSENCE OF AT LEAST ONE FIRST INHIBITION AREA|
    PCT/FR2016/051712| WO2017006055A1|2015-07-09|2016-07-06|Method for detecting a presence or absence of at least a first zone of inhibition|
    US15/743,148| US10870876B2|2015-07-09|2016-07-06|Method for detecting a presence or absence of at least one first zone of inhibition|
    EP16747827.0A| EP3320109B1|2015-07-09|2016-07-06|Method for detecting a presence or absence of at least a first zone of inhibition|
    CN201680050614.8A| CN108474019B|2015-07-09|2016-07-06|Method for detecting the presence or absence of at least one first inhibition zone|
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