Patent Application
20230175034
The technical field of the invention is characterization of microorganisms via an image acquired, in a transmission configuration, in the infrared.
There are many methods allowing colonies of microorganisms, cultivated in liquid nutrient media (broths) or on agar jelly, to be characterized. A first method consists in evaluating various visual or olfactory parameters of colonies. These parameters may be the shape of the colonies, color, odor or surface state. This method may be applied by an experienced operator, individually, i.e. colony by colony.
Certain automated and more accurate methods are destructive. They require samples of the colonies to be taken from their nutrient medium. It is for example a question of techniques based on mass spectrometry, such as MALDI-TOF mass spectrometry (MALDI-TOF standing for Matrix-Assisted Laser Desorption Ionization-Time of Flight). This is an effective method. However, it requires complex equipment and must be applied individually, to each colony. In addition, it is a destructive technique. Another potentially destructive technique used is Raman spectroscopy. This is also an individual technique that employs sophisticated equipment and requires a colony-by-colony analysis.
Multispectral imaging techniques may be used. It is a question of illuminating the colonies at various wavelengths, in the visible or ultraviolet or infrared spectral domain. In the visible, for example, bacterial colonies may be characterized on the basis of diffraction patterns.
When the nutrient medium is sufficiently transparent, the latter may be placed between a light source and an image sensor, in a transmission configuration. The image sensor may be used in a lensless configuration, i.e. with no image-forming optics between the nutrient medium and the image sensor. Such a configuration allows diffraction patterns to be obtained at one or more wavelengths. These form signatures, allowing the species of microorganisms present in the colonies to be identified. Examples of application are for example described in US20160103064 or in U.S. Pat. No. 7,465,560.
When the nutrient medium is opaque, it is not envisionable to use a transmission configuration. Applications employing backscattering have been described in US20190339199 or in US20190323959. The objective is to obtain an image representative of radiation backscattered by a bacterial colony. On account of geometrical constraints associated with the backscattering, this method is mainly limited to individual characterization of colonies.
In the infrared domain, application of transmission imaging is complicated by the fact that the nutrient media, on which the microorganisms develop, are absorbent. This is especially due to their high mass fraction of water, which may be higher than 80%.
The inventors have developed an optical method for characterizing microorganisms, in the infrared domain, in a transmission configuration: the characterized microorganisms are placed between alight source and an image sensor.
One subject of the invention is a method for characterizing microorganisms, comprising:
The nutrient medium contains nutrients propitious to the development of colonies of microorganisms. It may comprise a bactericide or a bacteriostat. It may also comprise an isotopic label, a chromogenic label, or a molecule that has a chemical bond that absorbs infrared light at a wavelength corresponding to the emission wavelength.
The characterization may be:
According to one embodiment
Preferably, the porous carrier transmits a least 1% of the light at the emission wavelength, or at each emission wavelength.
Step f) may comprise extracting a thumbnail from the image acquired in step e), the thumbnail being a region of interest of the acquired image corresponding to the colony of microorganisms. In step f), the thumbnail, or each thumbnail, may form an input datum of a supervised-learning algorithm, with a view to characterizing the microorganisms forming a colony.
According to one embodiment, step a) comprises seeding the porous carrier.
According to one embodiment, step a) comprises making a fluid, liable to contain microorganisms, flow through the porous carrier, from the first face to the second face, the carrier acting as a filter, so as to retain microorganisms on the first face, while permitting the fluid to flow through the pores. The fluid may be a liquid or a gas.
According to one embodiment, in step b), the nutrient medium:
According to one embodiment, in step b), the nutrient medium comprises a bactericide or a bacteriostat, an antibiotic for example, such that the image formed in step e), or each image formed in step e), is representative of a metabolic activity of the microorganisms when they are in contact with the nutrient medium.
According to one embodiment, following step a) and prior to step b), the method comprises an acquisition of an initial image of the porous carrier, such that metabolic activity may be determined via a comparison between the initial image and at least one image acquired in step e).
The thickness of the porous carrier, between the first face and the second face, is preferably smaller than 1 mm or than 500 μm. The diameter or largest diagonal of each pore may be comprised between 5 nm and 5 μm, and preferably between 20 nm and 500 nm. The porous carrier may be obtained from a material chosen from: alumina, silicon, germanium, zinc sulfide, silicon nitride, zinc selenide, a chalcogenide glass, calcium fluoride, and potassium bromide
The invention will be better understood on reading the description of examples of embodiment that are presented, in the remainder of the description, with reference to the figures listed below.
The diameter, or largest diagonal, of each face may for example be comprised between 1 cm and 10 cm, or more. The thickness ε of the porous carrier 15 is preferably smaller than 500 μm or than 1 mm or than 2 mm. It is preferably comprised between 10 μm and 1 mm, or between 10 μm and 500 μm. Depending on the thickness and on the material forming the carrier, the porous carrier 15 is flexible or rigid.
The porous carrier 15 comprises pores 153, extending from the first face 151 to the second face 152.
One of the functions of the porous carrier 15 is to form a carrier for microorganisms, and generally colonies of microorganisms 10i, placed on one of the faces. In the example shown, the microorganisms 10i are placed on the first face 151 of the carrier 15. The term microorganisms includes bacteria, archaea, microscopic fungi such as yeasts or filamentous fungi, microalgae, protozoa and parasites.
The diameter, or largest diagonal, of each pore 153, is preferably smaller than 5 μm, or even than 1 μm, or even than 500 nm, or even than 200 nm, and larger than 1 nm, or even than 5 nm, or even than 20 nm, or even than 50 nm. The diameter or largest diagonal is defined so as to allow microorganisms to be held on the first face 151 of the carrier.
The pores 153 allow the nutrient medium 17 to diffuse between the two faces of the carrier 15, while retaining microorganisms on one face of the carrier. The dimension of the pores 153 is chosen so as to be:
Thus, the microorganisms are retained on the first face 151, while developing by virtue of the provision of nutrients through the pores 153.
The light source 11 may be a light-emitting diode or a laser source. The light source 11 emits an incident light wave 12, at an emission wavelength λ. The emission wavelength λ is located in the spectral domain of the infrared. It may be a question of short wavelength infrared (SWIR), which extends between 1 and 3 μm, or of medium wavelength infrared (MWIR), which extends between 3 and 6 μm, or indeed of long wavelength infrared (LWIR), which extends between 6 and 20 μm. Thus, generally, the incident light wave 12 is emitted at a wavelength λ lying between 1 μm and 20 μm, this corresponding to a wave number comprised between 500 cm−1 10000 cm−1, or between 3 μm and 20 μm. The preferred spectral range is located between 3 μm and 11 μm, which corresponds to medium wavelength infrared, and to one portion of the long wavelength infrared. Specifically, this spectral range corresponds to the domain of the wavelengths of absorption of covalent bonds commonly encountered in organic molecules.
The light source 11 is preferably a laser source. It may especially be a wavelength-tunable laser source, for example a quantum cascade laser (QCL), and in particular an external-cavity laser. The width of the emission spectral bend of the light source is preferably smaller than 10 cm−1, or even than 5 cm−1, or even than 1 cm−1. The distance D between the light source and the carrier may be a few cm.
The light source 11 may comprise a plurality of elementary QCLs, respectively emitting in various spectral bands. The light source 11 may also be a black-body polychromatic source able to be associated with various bandpass filters defining the emission wavelength λ. Thus, the light source 11 may be polychromatic and wavelength-tunable.
The carrier 15 is placed between the light source 11 and the image sensor 20. The latter preferably lies parallel, or substantially parallel to one face of the carrier 15. The term substantially parallel means that the two elements may not be rigorously parallel, an angular tolerance of a few degrees, smaller than 20° or 10°, being acceptable. The carrier 15 may be placed on a holding element 19, configured to hold the carrier, between the light source 11 and the image sensor 20.
Under the effect of illumination by the incident light wave 12, which propagates along the propagation axis Z to the carrier 15, the latter transmits a light wave 14, which is called the transmitted light wave. The transmitted light wave 14 propagates, along the axis Z, to the image sensor 20. The carrier 15 and the microorganisms 10i may absorb one portion of the incident light wave 12. Thus, the transmitted light wave 14 corresponds to a portion of the incident light wave 12 that is not absorbed by the carrier 15 and by the colonies of microorganisms 10i.
The image sensor 20 is able to form an image of the transmitted light wave 14 in a detection plane P20. In this example, the image sensor is formed by a matrix array of pixels. Each pixel is an elementary infrared detector. Each pixel may for example be a bolometer, each bolometer of the matrix array having a detection spectral band comprised between 5 μm and 20 μm. The matrix array of bolometers may for example comprise 80×80 bolometers. According to variants, the image sensor may comprise a matrix array of pyrodetectors, or of photodiodes, the detection spectral band of which is located in the infrared, and preferably in the medium wavelength infrared. Thus, each pixel of the image sensor 20 may be an elementary infrared detector
The materials from which each pixel is formed may be chosen, nonlimitingly, from lead zirconate titanate (PZT), indium antimonide (InSb), mercury cadmium telluride (HgCdTe), lead sulfide (PbS), and lead selenide (PbSe).
The distance d between the image sensor 20 and the carrier 15 is preferably smaller than 5 mm. The smaller d is, the better the spatial resolution of the image acquired by the image sensor. Thus, it is advantage for the distance d to be smaller than 1 mm, or even smaller than 500 μm.
The field of observation of the image sensor 20 is defined by the size of the sensing area of the latter. The field of observation may be larger than 1 mm2, or even larger than 5 mm2 or 10 mm2. Thus, acquisition of a single image allows an intensity of the light wave 14 transmitted by an area of the carrier of several mm, and typically of at least 5 or 10 mm2, to be obtained simultaneously. Such a configuration allows simultaneous observation of a plurality of spatially separate colonies of microorganisms.
The holding element 19 may be movable, in particular parallel to the detection plane P20, so as to allow all or some of the surface of the carrier 15 to be scanned. It may in particular be a question of a motorized translation stage. The holding element 19 may also allow the carrier 15 to be rotated parallel to the detection plane.
A processing unit, for example taking the form of a microprocessor 21, is configured to perform, on the basis of the images acquired by the image sensor 20, image-interpreting operations that are described below. The microprocessor 21 is connected to a memory 22, which contains instructions relative to the image processing operations to be performed. It may also be connected to a screen 23.
One important element of the invention resides in the fact that the porous carrier 15 allows one portion of the incident light wave 12 to be transmitted to the image sensor 20. Preferably, its transmittance is higher than 0.01 (1%) or higher than 0.1 (10%). By transmittance, what is meant is a percentage of the light intensity transmitted by the material forming the porous carrier 15. It is advantageous for the transmittance to be as high as possible.
The material from which the porous carrier 15 is made may consist of or comprise a material having good transmission properties in the infrared. It may for example be a question of one of the following materials: alumina (aluminum oxide), silicon (Si), germanium (Ge), zinc sulfide (ZnS), zinc selenide (ZnSe), chalcogenide glasses based on sulfur, or on selenium or on telluride, silicon nitride (Si3N4), calcium fluoride (CaF2) or potassium bromide (KBr).
Use of the porous carrier 15 allows a detector 20 to be used in a transmission mode, the carrier 15 being interposed between the light source and the detector 20. This allows individual colonies of microorganisms to be characterized using the light wave 14 transmitted by the carrier 15 and collected by the detector 20. When the detector 20 is an image sensor, sensitive to infrared, each image Iλ acquired by the image sensor allows a characterization of the microorganisms. More precisely, each acquired image Iλ is representative of an absorption of the incident light wave 12 by the microorganisms, and by the porous carrier 15, at the wavelength λ.
It will be understood that the porous carrier 15 has a dual function:
Use of the porous carrier 15 allows the inability to form an image in transmission of microorganisms developing on nutrient media that are opaque in the infrared spectral domain to be overcome. Use of the porous carrier 15 allows the microorganisms to be moved between culture conditions, such as shown in
Moreover, the transmission-image-based analyzing method is non-destructive. It allows, following acquisition of an image, the carrier to be once again placed on a nutrient medium. The latter may be identical to or different from an initial nutrient medium, used prior to the image acquisition. It is thus possible to move the microorganisms between an initial nutrient medium and another nutrient medium different from the initial nutrient medium. This allows the ability of the microorganisms to develop in another nutrient medium to be evaluated, as described below.
As indicated above, the method allows a large field of observation to be addressed. Specifically, it is possible to place the carrier at a small distance from the image sensor 20, for example at a distance d smaller than 1 cm, or even to place it in contact with the image sensor. A large field of observation, the size of which is essentially dependent on the size of the image sensor, is thus achieved. Placing the image sensor at a sufficiently small distance d from the carrier makes it possible to avoid the need to use an image-forming optical system between the image sensor 20 and the carrier 15.
Apart from good transmittance properties in the infrared, the material from which the carrier 15 is formed is preferably not cytotoxic to the characterized microorganisms. It is preferably sterilizable, and compatible with conventional sterilization methods: sterilization by exposure to ionizing radiation (gamma radiation or x-rays for example) or non-ionizing radiation (microwaves or ultraviolet radiation), chemical sterilization (strong oxidants) or thermal sterilization (wet heat, dry heat, autoclave).
The formation of pores 153 in the material from which the carrier is formed may result from the application of a chemical method, electrochemical oxidation for example, or of a physical method, exposure to an electron beam or a beam of heavy ions for example. The method used to form the pores must allow through-pores 153 (i.e. pores that extend from the first face 151 to the second face 152) that are preferably of controlled size to be formed.
Two reference wavelengths (b8 and b9) have also been shown. The latter correspond to wavelengths that are considered not to be significantly absorbed by bacteria. Images formed at these wavelengths are representative of the absorbances of the carrier 15, and of the nutrient medium present in the pores 153, and of any gases present between the carrier and the image sensor, in particular CO2 or water vapor.
In
The pores, the size of which is distributed about an average value of 200 nm, may be seen to have a regular arrangement. The distribution of the size of the pores is considered to be uniform. In this example, porosity (volume fraction of the pores), measured by image processing, was estimated to be 59%. The higher the porosity, the higher the amount of potentially absorbent nutrient medium in the pores. The lower the porosity, the more difficult it is for the nutrient medium to reach the microorganisms. Generally, the porosity may be comprised between 0.1% and a few tens of %, for example 70% or 80%.
The inventors have devised a method for observing microorganisms, and more precisely colonies of microorganisms, the main steps of which are shown in
Step 100: placing microorganisms 10i on the first face 151 of the porous carrier 15. This step may consist in seeding the microorganisms using a spreading method, or in one of the variants described with reference to
Step 110: placing the porous carrier 15 in contact with a nutrient medium 17, such that the microorganisms may develop. Preferably, in this step, the second face 152 of the porous carrier 15 is placed on the free surface of the nutrient medium. In this step, the nutrient medium is contained in a chamber 16, a petri dish for example. As described above, the nutrient medium 17 diffuses through the pores 153 so as to reach the microorganisms 10i placed on the first face 151. This allows colonies to develop.
Step 120: removing the porous carrier 15 from the nutrient medium 17, and moving the porous carrier relative to the chamber 16. In this step, the porous carrier 15 is interposed between a light source 11 and an image sensor 20, such as described with reference to
Step 130: illuminating the carrier 15 at at least one wavelength λ, in the infrared domain, as described with reference to
Step 140: characterization
From the images of the stack of images Iλ1 . . . IλN, it is possible to extract thumbnails Ii,λ1 . . . Ii,λN, each thumbnail corresponding to a given colony observed in each image. Each thumbnail is a region of interest of an image, corresponding to a given colony. Examples of images, corresponding to various bacterial species will be described with reference to
Each thumbnail Ii,λ corresponds to a signature of the colony of microorganisms 10i at the wavelength Ii,λ. The signature depends:
The thumbnails Ii,λ allow the bacteria 10 to be identified. Characterization of the thumbnails thus allows the bacterial species forming the colony to be identified.
Step 140 may comprise identifying each colony on the basis of the thumbnails Ii,λ corresponding to said colony. To this end, the images are processed by a classifying algorithm, which is implemented by the processing unit 21. The processing algorithm may for example be an artificial-intelligence algorithm and for example a supervised-learning algorithm. It may for example be a SVM algorithm (SVM standing for support vector machine) or a neural-network algorithm or a random-forest algorithm. Use of a supervised-learning algorithm assumes a prior phase of training using images corresponding to known species of microorganisms.
Steps 100 to 140 were implemented using a carrier consisting of a membrane made of porous aluminum oxide: reference Anodisc (registered trademark)—manufacturer Whatman. The thickness of the carrier was 80 μm. The diameter of the pores was 200 nm. The carrier was sterilized by autoclave (pressure of 1 bar steam at 121.1° C. for 15 minutes). The carrier was placed on a solid nutrient medium, at 37° C., for an incubation time of 24 h. After incubation, the carrier was deposited directly on an image sensor, the bolometers of this image sensor being of 25 μm side length and of 37 μm pitch. The light source was a laser source tunable in steps of 1 cm−1, reference MIRcat—manufacturer Daylight Solutions, allowing an incident light wave of wavelength comprised between 5 μm and 11 μm to be emitted.
The inventors acquired thumbnails of 1012 colonies. The database thus formed was partitioned into 10 groups of equal size with a view to cross-validation. For each partition, 90% of the thumbnails (9 groups) were used for the purposes of supervised learning and 10% of the thumbnails (1 group) was used to test the performance of the classifying algorithm. Table 2 collates, in a confusion matrix, the percentage (between 0 and 1) of correct classifications in the test phases.
Candida
Escherichia
Enterobacter
Listeria
Staphyloccocus
albicans
coli
cloacae
innocua
epidermis
Candida
albicans
Escherichia coli
Enterobacter
cloacae
Listeria
innocua
Staphyloccocus
epidermis
Table 2 shows that the various species of microorganisms may be identified with a satisfactory confidence level. This attests to the relevance of the invention.
According to a first variant, the porous carrier 15 is also used as a filter, so as to retain microorganisms in a fluid medium, prior to their identification. This variant is illustrated in
Sub-step 101: mounting of the carrier.
Sub-step 102: filtration. This step is shown in
Sub-step 103: removal of the filter: the porous carrier 15 is removed so as to be placed on the surface of a nutrient medium 17.
Afterwards, the porous carrier 15 is deposited on the surface of the nutrient medium 17, the nutrient medium occupying a chamber 16 (see
The inventors have implemented the steps schematically shown in
An example of application of the first variant is to bacteriological analysis of water intended for human consumption. Alternatively, the fluid 31 is a gas. The invention allows microorganisms carried in a gas, air for example (industrial air, air in a hospital environment), to be retained and analyzed.
According to a second variant, the characterization of the microorganisms consists not in identifying them, but in evaluating their ability to develop. This allows indicators of sensitivity to a bactericide or a bacteriostat, an antibiotic for example, to be obtained. One example of an indicator is minimum inhibitory concentration (MIC), which corresponds to the minimum antibiotic concentration at which development of bacteria is inhibited.
Thus, the nutrient medium may contain an antibiotic with a known concentration, or with a known spatial concentration gradient, and for example a high concentration at its center and a low concentration on its periphery. Images of the carrier 15 may be taken at an initial time t and at a second time t2, respectively. Between the times t1 and t2, the carrier is placed on the surface of a nutrient medium containing an antibiotic. Comparison of the images obtained at the initial time t1 and at the second time t2, respectively, allows the development of the colonies between the two times to be evaluated. It is thus possible to characterize the ability of the analyzed microorganism to develop in the presence of the antibiotic.
According to one possibility, between the times t1 and t2, the nutrient medium contains, in addition to an antibiotic, an isotopic label present in a concentration higher than its natural concentration. The isotopic label is a molecule containing a stable isotope of an element. It may for example be a question of deuterium (D), substituted for hydrogen (H). Such a label is obtained using heavy water (D2O). The labelled nutrient medium may for example be a Mueller-Hinton medium containing heavy water, and a known concentration (or a known spatial concentration gradient). At the second time, the porous carrier 15 is illuminated at a wavelength absorbed by a chemical bond involving the isotope. It may for example be a question of the carbon-deuterium bond when the isotope is deuterium. If such a bond is revealed, by an image, to be present, it may be concluded that metabolism is ongoing in the labelled nutrient medium. Conversely, if the absence of such a bond is revealed it may be concluded that the bacteria is not metabolizing.
This variant corresponds to steps 100 to 130 and 150 to 170 shown in
In step 150, the porous carrier is placed in contact with a second nutrient medium 172 (see
In step 160, the porous carrier is removed from the second nutrient medium 172, then placed in an observing device such as shown in
In step 170, second images Iλ(t2) are formed at a second time t2 subsequent to the initial time t1. Comparison of the initial images Iλ(t1) and second images Iλ(t2) allows the ability of the microorganisms to metabolize in the second medium to be evaluated.
Instead of an isotopic label, a label containing a known covalent bond having an exploitable absorbance at an illumination wavelength of the light source may be used. It may for example be a question of the C≡N bond, which has an absorption wavelength at 2200 cm−1. Formation of images at said wavelength allows the metabolic activity of the microorganism in the second medium to be evaluated.
Instead of an isotopic label, a chromogenic medium able to induce a modification of the color of the microorganisms under the effect of their metabolism may be used.
The invention allows a high number of microorganisms to be characterized simultaneously and non-destructively. It does not require complex instrumentation and is particularly simple to implement. Furthermore, it is easily automatable.
| Number | Date | Country | Kind |
|---|---|---|---|
| FR2003377 | Apr 2020 | FR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2021/058664 | 4/1/2021 | WO |
