METHOD FOR DETECTING THE SENSITIVITY OF PHAGES OR ANTIBIOTICS TO BACTERIAL STRAINS

Description

Technical field of the invention

[0001] The present invention relates to a method for detecting the sensitivity of phages or antibiotics to a bacterial strain. State of the art

[0002] Phage therapy is a medical approach used to treat infectious diseases of bacterial origin, relying on the natural ability of certain viruses, called bacteriophages (and commonly referred to as phages), to destroy bacteria that they specifically recognize.

[0003] Implementing this approach requires first selecting phages that have a particular sensitivity for the bacteria to be destroyed. A classic method is to use a well plate, in which each well is filled with a suspension of bacteria. Phages of different composition and / or concentration are then added to each well. Each well is then monitored to see whether a lysis (destruction) reaction occurs or not. When lysis occurs in a well, it can be deduced that the phage added at the defined concentration has affinity and lytic activity for the bacteria. It will therefore be potentially usable for the implementation of phage therapy.

[0004] Patent application EP3822360A1 describes a solution using a principle for detecting lytic activity of phages by lensless imaging. A particular embodiment consists of placing a fluidic card with several chambers between a light source and an image sensor. In an exemplary embodiment, bacteria of the same bacterial strain are placed in each chamber and phages of distinct viral strains, or of the same viral strain but at distinct concentrations, are added. The reaction that occurs in each chamber is then observed. Using the image sensor, it is determined whether the bacteria is: Little or no sensitivity to the phage viral strain when the detected light intensity decreases between two successive measurement times, or Sensitive to the phage viral strain when the detected light intensity does not decrease or increases between two successive measurement times.

[0005] This previous solution may present risks of sample pollution, as each chamber may be exposed to the external environment.

[0006] Patent application WO2011 / 009213 relates to a method for testing affinities between biofilms and antimicrobial agents.

[0007] The aim of the invention is to propose a solution for detecting an interaction between a bacterial strain and bacteriophages or antibiotics of different composition / concentration which is: Automated or easy to automate; Simple and reliable to implement, allowing separate detection zones to be run in parallel, limiting the risk of cross-contamination, while not compromising bacterial growth; Quick to implement and allowing a large quantity of combinations to be tested, limiting the number of manipulations to be carried out;

[0008] This invention ultimately aims to identify, in a parallel manner, the types of phages with lytic activity against a targeted bacterium. The results thus produced then make it possible to define combinations of phage types ("phage cocktails"), likely to have therapeutic activity in vivo.

[0009] The invention thus makes it possible to determine the impact of various concentrations of a panel of antibacterials (antibiotics or bacteriophages) on the biomass of a bacterial strain. Statement of the invention

[0010] This aim is achieved by a method for detecting a lytic activity of one or more types of phages or the activity of one or more antibiotics with respect to a bacterial strain, the phages or antibiotics possibly being distinct from each other in composition and / or concentration, the method consisting of: Use a first multi-well plate, each well being made of a material transparent to light signals emitted by a light source and permeable to apolar gases, each well receiving a sample containing phages or antibiotics, Make at least one deposit on a transparent area of ​​a support, the deposit containing bacteria of the bacterial strain, Apply the first plate against the support so that each sample present in each well comes into contact with said deposit, Arrange the assembly formed by the first plate and the support between a light source and light detection means, Emit light signals through each well using the light source, For each well,determine using the detection means the activity or absence of activity of the phage or antibiotic with respect to said bacterial strain, taking into account the intensity of the light signals transmitted and / or its variation over time.

[0011] According to a particularity, the phages or antibiotics are present in each well in the form of a hydrogel.

[0012] According to another feature, the bacteria are dispersed in a gel deposited on said second plate so as to form said deposit.

[0013] According to another feature, the support is produced in the form of a second plate compartmentalized using watertight partitions so as to form several distinct compartments, said deposit being subdivided into several distinct deposits added in each compartment of the second plate.

[0014] According to another feature, the first plate is held against said second plate so that the phages or antibiotics contained in each well come into contact with a deposit placed in a separate compartment of the second plate. According to another feature, the first plate is turned over and moved to be applied by an open face on said wells against a functional face of the support carrying the deposit.

[0015] According to another feature, the support is returned so as to be applied by a functional face carrying said deposit against a face of the first plate open on said wells.

[0016] According to another feature, the method comprises a step of relative movement of the assembly formed by the first plate and the support relative to the detection means in order to transmit the light signals through each well of the first plate.

[0017] According to another feature, the detection means comprise an image sensor. According to another feature, the image sensor is configured to acquire a first image at a first time and a second image at a second later time and in that a processing unit is configured to determine that the bacterial strain is considered to be: Little or no sensitivity to phages or antibiotics when the intensity of the detected light signals decreases between the said two times, or Sensitive to phages or antibiotics when the intensity of the detected light signals does not decrease or increases between the said two times.

[0018] According to another special feature, the image sensor cooperates with the light source according to a lensless imaging principle.

[0019] According to another feature, the light source is configured to emit in a spectral band between 500nm and 600nm. Brief description of the figures

[0020] Other features and advantages will become apparent in the detailed description which follows, in conjunction with the attached figures listed below: There figure 1 schematically shows the detection system of the invention. The figure 2 illustrates the implementation of the detection method of the invention. Detailed description of at least one embodiment

[0021] The invention relates to a method which is suitable for: Detect the sensitivity, with respect to bacteria of the same bacterial strain, of phages from distinct viral strains; Detect the sensitivity, with respect to bacteria of the same bacterial strain, of phages from the same viral strain, at different concentrations; Detect the sensitivity, with respect to bacteria of the same bacterial strain, of phages from distinct viral strains and at different concentrations;

[0022] The same principle can be applied for antibiotics against bacteria of the same bacterial strain. In the remainder of the description, phages will be referred to in general terms, to discuss the detection principles of the method, defined above, but it should be understood that the invention can be applied in an identical manner to antibiotics.

[0023] The method of the invention makes it possible, on the same device, to bring B bacteria into contact with suspensions of P phages, distinct in composition and / or concentration, in a simple manner, by forming distinct detection zones.

[0024] The method is implemented using the detection system visible on the figure 1 .

[0025] This system comprises a first well plate 1. This first plate 1 thus comprises several juxtaposed wells 10. By "wells", we mean that the plate 1 comprises several distinct cavities, physically separated or sufficiently spaced from each other.

[0026] At the level of each of its wells, this plate 1 has the particularity of being made of a material: Transparent, preferably in the visible and / or near infrared, to allow light signals to pass through; Permeable to apolar gases, in particular to oxygen and carbon dioxide to guarantee bacterial growth; It will be seen that this characteristic is important because it makes it possible to guarantee bacterial growth even when implementing the method of the invention;

[0027] In a non-limiting manner, the first plate 1 is for example made entirely of a material such as PDMS (PolyDiMethyl Siloxane) which has the characteristics defined above. This material also has the advantage of being biocompatible.

[0028] According to the method of the invention, each well 10 is filled with a phage suspension. From one well to another, the phage suspensions P may be distinct in composition and / or concentration.

[0029] It should be noted that plate 1 may be presented in the form of a device pre-filled with P phages and thus ready for use. Plate 1 is, for example, kept sealed pending use.

[0030] The P phages can be dispersed in a hydrogel added in an appropriate amount to each well 10 of plate 1. This hydrogel can be a matrix made of agar-agar. Since this matrix is ​​composed of approximately 99% water, the P phages are stored in a saline aqueous environment, which helps to maintain the infectious titer of the phages over a long period.

[0031] Plate 1 may contain several dozen juxtaposed wells 10. This number may depend in particular on the size of the sensor 3 used for detection.

[0032] As indicated above, a material such as PDMS is impermeable to water but permeable to apolar gases, which allows gas exchange at the wells 10 and therefore ensures bacterial growth, even when the P phages and the B bacteria are brought into contact.

[0033] In the context of the invention, each well 10 thus materializes a delimited and distinct detection zone, through which the light source 4 can transmit light signals.

[0034] The system comprises a support for one or more deposits containing B bacteria of the bacterial strain. In a non-limiting manner and in the remainder of the description, this support may be formed of a second plate 2 capable of accommodating, over its entire surface or its functional volume, at least one deposit 20 of material. The support could also be formed directly by the surface of the sensor (see below).

[0035] The deposit 20 advantageously has sufficient thickness and viscosity to form a mat. This deposit 20 may be in the form of a gel containing the bacteria. It may be a gelatinous substance such as agar-agar, which is commonly used to cultivate bacteria. In a known manner, this substance is made from a red algae (compatible with the level of transparency required for detection) which provides a suitable growth surface for many varieties of bacteria. Of course, it would be possible to use another substance.

[0036] It should be noted that it is possible to produce a single deposit 20 covering the entire functional surface of the second plate 2. It is also possible to produce several juxtaposed deposits, each deposit being intended to be associated with a separate well of the first plate. To accommodate each of these deposits, the second plate 2 may have compartments, the compartments being partitioned in a sealed manner relative to each other. This compartmentalized version advantageously makes it possible to avoid any diffusion during the implementation of the method of the invention. It should indeed be noted that it is necessary to prevent the reaction localized at a well from spreading to an adjacent well, that is to say that phages contained in a well do not spread beyond their well, contaminating an adjacent well.

[0037] Each deposit 20 contains a non-zero concentration of B bacteria.

[0038] The second plate 2, as well as the deposit 20, are chosen to be transparent, preferably in the visible and / or near infrared, to the light signals L of the light source (see below) so as to be able to be crossed at least partially by these light signals L during the implementation of the method. In a non-limiting manner, the second plate can also be made of PDMS. But other materials such as PMMA ("Polymethylmethacrylate") or COC ("Cyclic Olefin Copolymer") could be considered.

[0039] The detection system comprises a light source 4. The light source 4 is for example composed of one or more light-emitting diodes or laser diodes. A bandpass filter (not shown - possibly integrated into the source) can be used at the output of the light source 4 or at the input of the image sensor 3, to adjust the spectral band of the light signals emitted. Preferably, the light signals L emitted by the light source 4 extend according to a spectral illumination band of the visible and near-infrared range. It is preferably between 400nm and 1000nm, preferably between 500nm and 600nm.

[0040] The detection system thus comprises an image sensor 3. This image sensor 3 may be a CCD or CMOS type sensor. In a non-limiting manner, it has, for example, a resolution of 5344x3516 pixels. It should be noted that the system advantageously does not comprise a magnification optic, the latter operating on the principle of lensless imaging.

[0041] The image sensor 3 is connected to a processing unit UC receiving the images acquired by the image sensor. The image sensor 3 is capable of generating several images at successive times, thus making it possible to monitor the evolution of the reactions at each well 10.

[0042] It would be possible to use other means of detection than the image sensor 3, for example a conventional microscope. The lensless imaging detection solution has the advantage of being able to monitor a large number of wells simultaneously, possibly without setting up complex mechanical means if all the elements of the system are correctly sized. It also allows simple image processing.

[0043] Starting from the different elements of the system as described above, the detection method is implemented in the manner described below, in conjunction with the figure 2. The first plate 1 is for example turned over and pressed, by its face open on the wells against the functional face of the second plate 2 carrying the deposit 20, so that the container of each well 10 comes into contact with the deposit 20 present on the functional surface of the second plate 2. The phages P present in each well 10 are thus brought into contact with the bacteria B inoculated in the deposit 20 of the second plate. The two plates 1, 2 are for example held against each other so as to create a seal from one well to another and to allow sufficient diffusion of the phages P in the deposit inoculated with bacteria B.

[0044] The permeability to apolar gases of the first plate 1 allows bacterial growth to be ensured, even when the P phages and the B bacteria are brought into contact, by applying the first plate 1 carrying the phages against the support of the B bacteria.

[0045] The assembly, for example formed of the two plates, is positioned between the light source 4 and the image sensor 3. It is for example positioned as close as possible to the surface 30 of the sensor 3, materialized for example by a protective glass, for example on this surface or just above. As already indicated above, it should be noted that the deposit 20 could be placed directly on the surface 30 of the sensor 3, the latter then serving as a support for the bacteria B.

[0046] The light source 4 is activated so as to illuminate the different wells 10. The lighting can make it possible to illuminate all the wells 10 simultaneously or one after the other or in groups of several wells. In the latter case, actuation means can be provided to effect a movement of the assembly formed by the two plates and / or the light source 4.

[0047] The image sensor 3 is activated so as to capture, simultaneously or almost simultaneously, images of each well 10.

[0048] In the absence of phages or notable effects of P phages on the bacterial strain, the bacteria proliferate. When P phages exhibit a particular sensitivity to the bacterial strain and a lytic activity on it, the number of B bacteria decreases. The increase or decrease in the number of B bacteria at each well 10 then results in a variation in the nature and / or intensity of the light transmitted through each well 10. The images acquired by the sensor 3 over time thus make it possible to assess and quantify this evolution. In other words, at each well 10, if the P phage exhibits a particular lytic activity with respect to the bacterial strain, it will tend to lyse the B bacteria. The intensity of the light emitted by the light source 4 through the well 10 and captured by the image sensor will increase.

[0049] On the other hand, if the phage P present in said well only has a weak lytic activity, or even no lytic activity for the bacterial strain, the intensity of the light emitted by the light source 4 through the well 10 and captured by the image sensor 3 will be stable or will decrease, due to the proliferation of the bacteria B. The detection principles are for example described in the patent application EP3822360A1 .

[0050] On the figure 2 , we can thus see that, at the level of each well 10, the phages diffuse into the deposit 20 inoculated with B bacteria. Certain P phages which present a sensitivity with the bacterial strain come to destroy the B bacteria and others which do not have it will cohabit with the B bacteria, the B bacteria then being able to continue their proliferation. On this figure 2, the P phages initially present in wells 10_2, 10_4, 10_5 show sensitivity to the bacterial strain and lytic activity on the bacteria. The light signals transmitted through these wells are of a higher intensity than those transmitted through the other wells 10_1, 10_3, 10_6, for which the P phages (in composition and / or concentration) do not show a particular sensitivity to the bacterial strain. In wells 10_1, 10_3, 10_6, the light signals are in fact attenuated by the compounds (bacteria + phages) present.

[0051] The same principle applies with a second compartmentalized plate.

[0052] To image all the wells, it should be noted that it is also possible to move the image sensor 3 and / or the first plate / second plate assembly, if it turns out that the capture surface 30 is not sufficient to view all the wells 10 in a single image.

[0053] The invention allows rapid testing of the lytic activity of P phages against a bacterial strain. The test can be performed in just a few hours.

[0054] For example, using an 864mm2 image sensor, it was possible to monitor sixty-six wells simultaneously. Of course, it would be possible to increase the number of wells on a single plate, and / or increase the sensor surface area and / or even use several juxtaposed sensors.

[0055] It should also be noted that the principle of the invention makes it possible to monitor in real time the lytic activity of phages on bacteria. The kinetic information from real-time acquisition thus provides an additional layer of information.

[0056] The invention has many advantages, including: Multiplexing of phage activity measurements on bacterial strains, The use of conventional components such as well plates, image sensors, light sources, A process that is easy to implement and capable of providing results quickly, A reliable process as it requires little handling, A process that allows continuous monitoring, which can provide faster results when implemented in an incubator.

Claims

1. Method for detecting a lytic activity of one or more types of phages (P) or of the activity of one or more antibiotics with respect to a bacterial strain, the phages (P) or antibiotics optionally being distinct from each other in terms of composition and / or concentration, characterized in that it consists in: - using a first multiwell plate (1), each well (10) being made of a material transparent to the light signals (L) emitted by a light source (4) and permeable to apolar gases, each well (10) receiving a sample containing phages or antibiotics, - making at least one deposit (20) on a transparent area of a support, the deposit (20) containing bacteria (B) of the bacterial strain, - applying the first plate (1) against the support so that each sample in each well (10) comes into contact with said deposit (20), - placing the assembly formed by the first plate (1) and the support between a light source (4) and light detection means, - emitting light signals (L) through each well (10) using the light source (4), - for each well, determining, using the detection means, the activity or absence of activity of the phage or antibiotic with respect to said bacterial strain, taking into account the intensity of the transmitted light signals and / or the variation thereof over time.

2. Method according to Claim 1, characterized in that the phages or antibiotics are present in each well in the form of a hydrogel.

3. Method according to Claim 1 or 2, characterized in that the bacteria (B) are dispersed in a gel deposited on said second plate (2) so as to form said deposit (20).

4. Method according to one of Claims 1 to 3, characterized in that the support is made in the form of a second plate (2) compartmentalized by means of leaktight partitions so as to form several distinct compartments, said deposit being subdivided into several distinct deposits added to each compartment of the second plate.

5. Method according to Claim 4, characterized in that the first plate (1) is held against said second plate (2) so that the phages (P) or antibiotics contained in each well come into contact with a deposit placed in a compartment separate from the second plate.

6. Method according to one of Claims 1 to 5, characterized in that the first plate (1) is turned upside down and moved so as to come to bear, via an open face on said wells (10), against a functional face of the support carrying the deposit (20).

7. Method according to one of Claims 1 to 5, characterized in that the support is turned upside down so as to come to bear, via a functional face carrying said deposit (20), against a face of the first plate (1) open on said wells (10).

8. Method according to one of Claims 1 to 7, characterized in that it comprises a step of relative displacement of the assembly formed by the first plate (1) and the support relative to the detection means for the purpose of transmitting the light signals (L) through each well (10) of the first plate (1).

9. Method according to one of Claims 1 to 8, characterized in that the detection means comprise an image sensor (3).

10. Method according to Claim 9, characterized in that the image sensor (3) is configured to acquire a first image at a first time and a second image at a later second time and in that a processing unit is configured to determine that the bacterial strain is considered to be: - barely or not at all sensitive to the phages or antibiotics when the intensity of the detected light signals decreases between said two times, or - sensitive to the phages or antibiotics when the intensity of the detected light signals does not decrease or increases between said two times.

11. Method according to Claim 9 or 10, characterized in that the image sensor (3) cooperates with the light source (4) according to a lensless imaging principle.

12. Method according to one of Claims 1 to 11, characterized in that the light source is configured to emit in a spectral band between 500 nm and 600 nm.