FR3110574A1 - CAPTURE OF BACTERIA USING BACTERIOPHAGES OR BACTERIOPHAGE PROTEINS - Google Patents

Abstract

CAPTURE OF BACTERIA USING BACTERIOPHAGES OR BACTERIOPHAGE PROTEINS The present invention relates to the use of a solution comprising a cation and / or a protein, for the capture and / or for the detection and / or quantification of a bacterium by a bacteriophage or bacteriophage protein, said bacteriophage protein preferably being a receptor binding protein (RBP) and said solution preferably comprising a cation and a protein. Abstract figure: Fig. 1

Description

CAPTURE OF BACTERIA USING BACTERIOPHAGES OR BACTERIOPHAGE PROTEINS

FIELD OF THE INVENTION

The present invention relates to a device and a method for the rapid detection of bacteria in a sample.

STATE OF THE ART

Bacterial infections are responsible for many pathologies, especially in farms and more particularly in sheep or cattle farms.

For this, it is essential to be able to detect the presence of the bacterium responsible for the infection, particularly in the breeding environment, and more particularly rapid detection methods in order to treat and prevent bacterial infections. However, to date, no detection method is fully satisfactory.

For example, diagnostic devices on the market, in particular mastitis detection devices, are mainly based on the detection of somatic cells found in milk. However, this method does not make it possible to identify the bacterium(ies) responsible for the infection. On the other hand, the latter can constitute a trace of a more or less old infection and therefore constitute a non-specific indicator of an ongoing infection. This therefore leads breeders to use antibiotic therapies on a massive and non-targeted basis.

Other bacteria detection methods can be used such as the polymerase chain reaction (PCR) or the culture method. However, these require sending the samples to the laboratory and therefore the intervention of qualified people to implement the detection method and to analyze the results.

In addition, the use of the culture method requires an incubation time of 24 to 48 hours, which represents too long a wait for results, given the challenges of the sector. Regarding the molecular biology approach (PCR and/or quantitative PCR), although faster and more specific, it is much more expensive. In addition, the PCR technique only detects the presence of bacterial DNA and not the presence of viable bacteria, which can lead to the detection of false positives (dead bacteria and already taken care of by the host's immune system) .

These methods therefore have the disadvantages of being long, tedious, of using chemical compounds that are often toxic for the operator and for the environment, of requiring expensive equipment, such as extractor hoods, protective gloves, centrifuges , etc., and to be implemented in suitable premises, such as laboratories. They do not meet the needs of field users, especially livestock farmers.

As a particularly illustrative example, the case of the veterinarian wishing to quickly identify an infectious pathogenic agent directly at the location of the sick animal can be cited. This so-called “point-of-care testing” (POCT) approach is the subject of particular attention from the professionals concerned.

There is therefore a need to provide means allowing rapid, inexpensive detection of bacteria, which is not harmful to the operator and to the environment, and does not require the constraint of implementation in a dedicated laboratory.

There is also a need to provide means allowing “universal” detection of bacteria, regardless of the nature of the original sample containing said bacteria.

Such a method for detecting bacteria could also be applied in various fields such as, for example, in food safety, water control or in human health.

SUMMARY

The invention therefore relates to the use of a solution comprising a cation and/or a protein, for the capture and/or for the detection and/or the quantification of a bacterium by a bacteriophage or bacteriophage protein, said protein of bacteriophage preferably being a receptor binding protein (RBP) and said solution preferably comprising a cation and a protein.

The invention also relates to the use of a solution for the capture and/or for the detection and/or the quantification of a bacterium by a bacteriophage or bacteriophage protein comprising a cation and/or a protein in which the protein is a milk protein in particular albumin, even more particularly bovine serum albumin (BSA), a serum, more particularly chicken serum or an antibody.

In another implementation, the solution as used comprises a cation chosen from cations containing a divalent cation or dication, in particular Mg ²⁺ or Ca ²⁺ or even containing Na ⁺ , K ⁺ , Fe ²⁺ , Fe ³⁺ , K ⁺ , Zn ⁺ , Au ³⁺ , Cu ⁺ , Carbocation, Mn ⁺ , Ag ⁺ , more particularly comprising the salts MgSO ₄ , Na ₂ SO ₄ , MgCl ₂ , CaCl ₂ , CaSO ₄ , K ₂ SO ₄ or KCl.

In a particular implementation, the solution containing a cation and a protein is such that the cation concentration, in particular MgSO ₄ , is between 5 mM and 300 mM inclusively and the protein concentration, in particular BSA, is between 0.1 mg/mL to 5 mg/mL inclusive.

In another particular implementation, the solution containing a cation and a protein is used for the fixing between Staphylococcus aureus a bacteriophage is specific for Staphylococcus aureus .

In a particular implementation, said bacteriophage is chosen from bacteriophages specific for S. aureus as deposited at the CNCM under the numbers CNCM I-5408, CNCM I-5409, CNCM I-5410, CNCM I-5491 and CNCM I- 5492, or is a mixture of these bacteriophages

In another implementation, the bacteriophage protein is a receptor-binding protein (RBP), in particular specific to Staphylococcus aureus , more preferably chosen from an RBP of bacteriophage CNCM I-5408, in particular an RBP of sequence SEQ ID NO:1, an RBP from bacteriophage CNCM I-5409, in particular an RBP of sequence SEQ ID NO:2, an RBP from bacteriophage CNCM I-5410, in particular an RBP of sequence SEQ ID NO: 3, or a mixture of these.

Another aspect of the invention relates to a process for preparing a device comprising a solid support, said device preferably being a microtiter plate or a strip, said process comprising:

a step a) of coupling a bacteriophage or of a combination of bacteriophages or of a protein of bacteriophages or of a combination of proteins of bacteriophages on the solid support, the said coupling step consisting in bringing the support into contact with a bacteriophage or a combination of bacteriophages and/or a bacteriophage protein or a combination of bacteriophage proteins in the presence of a solution comprising a cation and/or a protein, the bacteriophage protein preferably being an RBP, the method optionally comprising a step b) of eliminating the bacteriophage(s) and/or the bacteriophage protein(s) not bound to the solid support, and optionally a step c) of rinsing, which can be repeated at least once.

In a particular implementation, the device obtained according to the preparation process of the invention is preferably a microtiter plate or a strip and the support being preferably made of polystyrene, polypropylene, polycarbonate, cellulose acetate, cellulose, glass, and/ or silicon.

In another aspect, the invention relates to a device chosen from a microtitration plate or a strip comprising a solid support on which is fixed at least one receptor-binding protein (RBP), the support being preferably made of polystyrene, polypropylene, polycarbonate, cellulose acetate, cellulose, glass, and/or silicon, the support is in particular precoated with at least one protein.

In another aspect, the subject of the invention is the use of the device according to the invention for the detection and/or the quantification of one or more bacteria present in a sample.

In another aspect, the invention relates to a method for the detection and/or quantification of one or more bacteria present in a sample with a device of the strip or ELISA type, comprising the following steps

1. bringing the sample likely to contain bacteria into contact with a device according to the invention,
2. optionally elimination of bacteria not related to bacteriophages and / or bacteriophage proteins,
3. detection of bacteria bound to bacteriophages and/or bacteriophage proteins with a bacterium-specific ligand.

In a particular implementation, the method for the detection and/or the quantification of one or more bacteria present in a sample in which the ligand specific for the bacterium of step c) comprises a bacteriophage specific for the labeled bacterium, a bacteriophage protein specific for the labeled bacterium, a bacteriophage specific for the bacterium coupled to a labeled antibody or a bacteriophage protein coupled to a labeled antibody, and in which the said step c) is carried out in the presence of a solution comprising a cation and/or a protein, preferably a cation and a protein, or the bacterium-specific ligand is a labeled antibody or a labeled aptamer directed against the bacterium.

In a particular aspect, the subject of the invention is a method for the detection and/or quantification of one or more bacteria present in a sample with an immunodetection device of the strip or ELISA type, comprising the following steps :

1. bringing the sample likely to contain bacteria into contact with an immunodetection device, on which is fixed a ligand specific for the bacteria, preferably an antibody specific for the bacteria or an aptamer specific for the bacteria,
2. optionally elimination of bacteria not bound to the specific ligand of the bacterium,
3. detection of bacteria bound to the bacterium-specific ligand using a solution comprising a labeled bacteriophage specific to the bacterium, a labeled bacteriophage protein specific to the bacterium, a bacteriophage specific to the bacterium coupled to a labeled antibody or a bacteriophage protein coupled to a labeled antibody, and a cation and/or a protein, preferably a cation and a protein

The invention also relates to a receptor-binding protein (RBP) of a bacteriophage specific for S. aureus , said RBP being chosen from an RBP of bacteriophage CNCM I-5408, in particular an RBP of sequence SEQ ID NO: 1, an RBP from bacteriophage CNCM I-5409, in particular an RBP of sequence SEQ ID NO: 2, and an RBP from bacteriophage CNCM I-5410, in particular an RBP of sequence SEQ ID NO: 3.

DEFINITIONS

In the present invention, the terms below are defined as follows:

A “bacteriophage specific for a bacterium” is a bacteriophage capable of binding specifically to this bacterium. This specificity can be determined by the spot method described in the present application. It is in particular a bacteriophage specific for S. aureus , more particularly, a bacteriophage chosen from the bacteriophages specific for S. aureus as deposited at the CNCM under the numbers CNCM I-5408, CNCM I-5409 , CNCM I-5410, CNCM I-5491 and CNCM I-5492. In particular, the bacteriophage specific for S. aureus is a mixture of bacteriophages specific for S. aureus chosen from the strains deposited at the CNCM under the numbers CNCM I-5408, CNCM I-5409, CNCM I-5410 CNCM I-5491 and CNCM I-5492.

“Bacteriophage” and “phage” are used interchangeably and refer to the same entity.

Bacteriophage protein specific to a bacterium is a protein capable of binding specifically to this bacterium.

“X conjugate to Y” and “Y conjugate to X” are used interchangeably.

“Bivalent cation” and “dication” are used interchangeably.

“Phage-containing solution” and “phage-containing suspension” are used interchangeably.

A “bacterium-specific ligand” is a ligand capable of binding specifically to bacteria. This specificity can be determined by the method of magnetic beads for example.

A “conjugated label” can be a colloidal molecule, for example: a gold nanoparticle (AuNP) or colloidal gold, a carbon nanoparticle or colloidal carbon, a colored latex ball, a magnetic particle, or a fluorescent molecule, a molecule luminescent agent or an enzyme such as horseradish peroxidase which catalyzes the conversion of chromogenic substrates into colored compounds, or which produces light from chemiluminescent substrates.

A “marked” element is an element (protein, bacterium, molecule) conjugated to a molecule that can be detected visually. The marker is used to label a bacterium.

A “surfactant” is an amphiphilic molecule that lowers the surface tension of an aqueous solution. For example, the surfactant is Tween 20.

A “strip” refers to a detection device Lateral flow chromatography also known as lateral flow immunochromatography is a method that relies on the movement of a liquid sample by capillary action through a strip. It has previously been described for the immunological detection of an analyte of interest in a liquid sample, using a primary antibody and a secondary antibody (EP1086372B1). They are simple paper-based devices intended to detect the presence (or absence) of a target analyte in a liquid sample (matrix).

A "microtritration plate" refers to a device comprising a set of wells, it is a standard tool in research and clinical analysis of laboratory diagnostic tests. Their use is very frequent in the Enzyme-linked immunosorbent assay (ELISA).

DETAILED DESCRIPTION

The present invention relates to the use of a solution comprising a cation and/or a protein, for the capture and/or for the detection and/or the quantification of a bacterium by a bacteriophage or bacteriophage protein, said bacteriophage protein preferably being a receptor binding protein (RBP) and said solution preferably comprising a cation and a protein.

In the context of the present invention, the bacteriophages or the bacteriophage proteins are in solution, said solution comprising a cation and/or a protein, preferably a cation and a protein.

The use of such a solution comprising a cation and/or a protein, preferably a cation and a protein. The use of the solution makes it possible to increase the capture of a bacterium by the phage or the phage protein.

The solution can be used for the procedure of capture, detection and/or quantification of a bacterium by bacteriophages or by bacteriophage protein.

The protein of this solution is chosen in particular from milk proteins, in particular albumin, even more particularly bovine serum albumin (BSA), or a serum protein, more particularly chicken serum, or else an antibody.

In a particular embodiment, the cation is chosen from a divalent cation, in particular Mg ²⁺ or Ca ²⁺ or else containing Na ⁺ , K ⁺ , Fe ²⁺ , Fe ³⁺ K ⁺ , Zn ^+, Au ³⁺ , or also Cu ^+, carbocation, Mn ⁺ , Ag ⁺ , more particularly comprising the MgSO ₄ , Na ₂ SO ₄ , MgCl ₂ , CaCl ₂ , CaSO ₄ , K ₂ SO ₄ or KCl salts.

In a particular embodiment, the cation concentration in the solution is between 5 mM and 300 mM inclusive. In a more particular embodiment, the solution comprises MgSO ₄ whose concentration is between 5 mM and 300 mM inclusively.

In a particular embodiment, the protein concentration in the solution is between 0.1 mg/mL to 5 mg/mL inclusive. In an even more particular embodiment, the protein is BSA and its concentration is between 0.1 mg/mL to 5 mg/mL.

In one embodiment, the solution comprises BSA at a concentration of 0.1 mg/mL to 5 mg/mL and MgSO ₄ at a concentration of 5 mM and 300 mM inclusively.

In a particular embodiment, the solution comprises BSA at a concentration of 2 mg/mL and MgSO ₄ at a concentration of 50 mM.

In a particular embodiment, said composition also comprises a stabilizing agent, such as glycerol and/or a sugar, for example sorbitol and/or sucrose.

In the context of the present invention, any type of bacteriophage specific for a bacterium can be used. It is in particular a bacteriophage specific for S. aureus, more particularly, a bacteriophage chosen from bacteriophages specific for S. aureus as deposited at the CNCM under the numbers CNCM I-5408, CNCM I-5409 , CNCM I-5410, CNCM I-5491 and CNCM I-5492. In particular, the bacteriophage specific for S. aureus is a mixture of bacteriophages specific for S. aureus chosen from the strains deposited at the CNCM under the numbers CNCM I-5408, CNCM I-5409, CNCM I-5410 CNCM I-5491 and CNCM I-5492

In the context of the present invention, any type of bacteriophage protein specific to a bacterium can be used. This is in particular a phage receptor binding protein (Receptor Binding Protein or RBP; RBP). It is in particular a protein of a bacteriophage specific for S. aureus, said bacteriophage specific for S. aureus being in particular chosen from bacteriophages specific for S. aureus as deposited at the CNCM under the numbers CNCM I -5408, CNCM I-5409, CNCM-I-5410, CNCM I-5491 and CNCM I-5492. More particularly, it is an RBP of bacteriophage CNCM I-5408, in particular an RBP of sequence SEQ ID NO: 1, an RBP of bacteriophage CNCM-I-5409, in particular an RBP of sequence SEQ ID NO: 2, an RBP of bacteriophage CNCM I-5410, in particular an RBP of sequence SEQ ID NO: 3. In particular, said protein of a bacteriophage specific for S. aureus is a mixture of RBPs of bacteriophages specific for S. aureus , said bacteriophages specific for S. aureus being chosen from the strains deposited at the CNCM under the numbers CNCM I-5408, CNCM I-5409, CNCM I-5410 CNCM I-5491 and CNCM I-5492. For example, it is a mixture of an RBP of bacteriophage CNCM I-5408 and an RBP of bacteriophage CNCM I-5409, an RBP of bacteriophage CNCM I-5408 and an RBP of bacteriophage CNCM I-5410, an RBP of bacteriophage CNCM-I-5409 and an RBP of bacteriophage CNCM I-5410 or an RBP of bacteriophage CNCM I-5408, an RBP of bacteriophage CNCM I-5409 and of an RBP of bacteriophage CNCM I-5410. More particularly, it is a mixture of an RBP of sequence SEQ ID NO: 1 and an RBP of sequence SEQ ID NO: 2, an RBP of sequence SEQ ID NO: 1 and an RBP of sequence SEQ ID NO: 3, of an RBP of sequence SEQ ID NO: 2 and of an RBP of sequence SEQ ID NO: 3, or of an RBP of sequence SEQ ID NO: 1, of an RBP of sequence SEQ ID NO: 1 and an RBP of sequence SEQ ID NO: 3.

In the context of the present invention, the use of the solution comprising a cation and/or a protein is implemented in the presence of a support. In particular, in the context of the detection procedure according to the invention, the bacteriophages or bacteriophage proteins are immobilized on supports, for example microtiter plates, test strips, slides, platelets, filter materials or continuous flow cell chambers. The support structures can for example comprise polystyrene, polypropylene, polycarbonate, PMMA, cellulose acetate, nitrocellulose, glass or a silicon wafer.

In the context of implementation on a tape-type medium, the medium is a paper-based device.

The subject of the present invention is a method for preparing a device comprising a solid support, said device preferably being a microtiter plate or a strip, said method comprises a step a) of coupling a bacteriophage or a combination of bacteriophages or a bacteriophage protein or a combination of bacteriophage proteins on the solid support, said coupling step comprising contacting the support with a bacteriophage or a combination of bacteriophages or a bacteriophage protein or a combination of bacteriophage proteins in the presence of a solution comprising a cation and/or a protein, the bacteriophage protein preferably being an RBP, optionally a step b) of eliminating the bacteriophage(s) or the uncoupled bacteriophage protein(s)/ bound to the solid support, and optionally a step c) of rinsing, which can be repeated at least once.

In a particular implementation, the subject of the invention is a method for preparing a strip comprising a step a) of coupling a bacteriophage or a combination of bacteriophages or a bacteriophage protein or a combination of bacteriophage proteins on said strip comprising a solid support, said coupling step comprising contacting the support with a bacteriophage or a combination of bacteriophages or a protein of bacteriophages or a combination of bacteriophage proteins in the presence of a solution comprising a cation and a protein, the bacteriophage protein preferably being an RBP, optionally a step b) of eliminating the bacteriophage(s) or the bacteriophage protein(s) not coupled/bound to the solid support, and optionally a step c) rinsing, which can be repeated at least once.

One of the objects of the present invention is a strip obtained by the method described above, thus said strip comprises a bacteriophage or a combination of bacteriophages or a protein of bacteriophages or a combination of proteins of bacteriophages attached to its surface of said strip and more particularly on the surface of the conjugate pad or of the area for detecting the bacterium of interest of the nitrocellulose membrane located after the conjugate pad. These implementations are more particularly described in the examples below.

Another object of the invention is a microtiter plate obtained by the method described above using a solution comprising a cation or a protein, thus said microtiter plate comprises a bacteriophage or a combination of bacteriophages or a bacteriophage protein or a combination of bacteriophage proteins attached to its surface ( ie , surface of the solid support). In a particular implementation, the microtiter plate is obtained by the method described above using a solution comprising a cation and a protein, thus said microtiter plate comprises a bacteriophage or a combination of bacteriophages or a bacteriophage protein or a combination of bacteriophage proteins attached to its surface ( ie , surface of the solid support).

In another implementation according to the invention, the device obtained according to the preparation process is a microtiter plate or a strip and the support being preferably made of polystyrene, polypropylene, polycarbonate, cellulose acetate, cellulose, glass, and/or silicon.

In a particular implementation of the invention, the device according to the invention is a microtiter plate or a strip which comprises a solid support on which is fixed a receptor-binding protein (RBP), the support preferably consisting of polystyrene, polypropylene, polycarbonate, cellulose acetate, cellulose, glass, and/or silicon, the support is in particular precoated with a protein.

The immobilization, capture or binding of bacteriophage or bacteriophage protein is one aspect of the invention. What is important in this regard is the functional immobilization, i.e. that the bacteriophages or bacteriophage proteins, antibodies or aptamers have structures accessible to bacteria, despite binding to the support material.

To eliminate a non-specific reaction of the bacteria to be studied with the material of the support, it is possible to implement in particular prior to the coupling or after the coupling on a solid support, a blocking with a protein, in particular bovine serum albumin or Tween 20 or known similar substances, such as powdered milk or an antibody that is not specific to the bacteria.

Immobilization can be obtained on a bare support or on a support precoated with a protein and in particular albumin or an antibody.

These media can be produced prior to their use and be stored for their use.

To this end, a solution comprising a cation and/or a protein and bacteriophages or bacteriophage proteins, in particular RBP proteins, is applied to the support. After adsorption, for example at 4-20°C overnight or at 30-65°C for 2-4 hours, the solution is removed and the support structure is dried.

According to a particular aspect, the invention relates to a chromatography strip for detecting a bacterium of interest in a sample.

The particularity of the detection strip of the invention is the blocking of the migration of bacteria on the support.

Sling embodiments are depicted in Figures 1 through 6.

The strip embodiments described below are described before use of said strip, that is to say before contacting with the sample to be analyzed.

Thus, the present invention relates in particular to a strip for detecting a bacterium of interest, in particular S. aureus , in a sample, comprising at least one part which comprises at least one bacteriophage specific for the bacterium of interest and/or at least a protein from a bacteriophage specific for the bacterium of interest.

In a particular embodiment, said lateral flow chromatography strip comprises in order at least:

• a contact part (sample pad) comprising a contact zone with the sample;
• a detection part comprising a zone for detecting the bacterium of interest which comprises at least one bacteriophage specific for said bacterium of interest and/or at least one protein of at least one bacteriophage specific for said bacterium of interest,
• an absorbent part.

The contact part (sample pad) comprises a contact zone intended to be brought into contact with the sample. Its function is to evenly distribute the sample so that the migration of the sample by capillarity to the following parts is smooth, continuous and homogeneous. The contact part and the contact zone are a capillary matrix. For example, the contact part and the contact area are made of cellulose or fiberglass.

The labeling part (conjugate or conjugate pad) includes a labeling area where the bacteria are labeled with a marker when the flow of sample to be analyzed passes through it. Their role is to maintain the marker until the strip is used and to release the marker when there is recognition between the marker and a bacterium, when the sample flow passes. For example the marking part and the marking area are made of fiberglass, cellulose or polyester

The detection part (detection pad) includes a detection zone where the bacterium of interest, if it is present in the sample analyzed, is captured on the strip thanks to a ligand specific for it which is immobilized there. The role of the detection part is therefore to provide a good support and a good bond for the immobilization of specific ligands, while limiting non-specific adsorptions. For example, the detection part and the detection zone are a nitrocellulose membrane.

Within the meaning of the present application, a ligand specific for a bacterium is chosen from an antibody directed against the bacterium, a bacteriophage specific for the bacterium, a protein of a bacteriophage specific for the bacterium, a bacteriophage specific for the bacterium coupled to a antibody directed against the bacterium, a bacteriophage protein specific for the bacterium coupled to an antibody directed against the bacterium In particular, it is a specific ligand for S. aureus and is therefore selected from an antibody directed against S . aureus (or anti- S . aureus ), a bacteriophage specific for S. aureus , a protein of a bacteriophage specific for S. aureus , a bacteriophage specific for S. aureus coupled with an anti- S . aureus , a bacteriophage protein specific for S. aureus coupled to an anti- S . aureus .

The absorbent part (absorbent pad) has an absorption role. It thus helps to maintain the flow rate through the strip and allows the recovery of excess sample and reagents, thus avoiding sample reflux. For example, the absorbent part is made of cotton.

Within the meaning of the present application "in order" means the order in which the different parts which make up the strip will be crossed by the flow containing the sample, but this term does not mean that these parts are necessarily contiguous.

In a particular embodiment, one of the ends of the contact part is superimposed with one of the ends of the part downstream of the latter to form a first zone of superposition, one of the ends of the part of detection is superimposed with one of the ends of the part upstream of the latter to form a second overlapping zone and the other end of the detection part is superimposed with one of the ends of the part downstream of the latter to form a third superposition zone, and one of the ends of the absorbent part is superposed with one of the ends of the part downstream thereof to form a fourth superposition zone. In particular, the first, the second and the third overlap zone are distinct from each other. In particular, the contact zone is distinct from the first overlapping zone. In particular, the detection zone is distinct from the second and from the third superposition zone.

In a particular embodiment, one of the ends of the detection part is superimposed with one of the ends of the contact part and the other end of the detection part is superimposed with one of the ends of the absorbent part.

In a more particular embodiment, one of the ends of the detection part is superimposed with one of the ends of the contact part to form a first overlapping zone, and the other end of the detection part is superimposed with one of the ends of the absorbent part to form a second overlapping zone. In particular, the first, the second and the third overlap zone are separated from each other. In particular, the contact zone is distinct from the first overlapping zone. In particular, the detection zone is distinct from the first and from the second superposition zone.

In a particular embodiment, the detection zone is an area where the phages/phage proteins are deposited (Figures 1, 3).

In a particular embodiment, the detection zone is distributed after the sample deposit zone (sample pad) and after the conjugate zone (conjugate pad) if it is present ( ) or if it is absent ( ) but before the adsorption zone

In a particular embodiment, the detection zone merges with the conjugate zone (conjugate pad) ( and 6).

In a particular embodiment, said strip further comprises, between the contact part and the detection part, a marking part which comprises a marking zone comprising a marker.

The labeling part (conjugate pad) includes a labeling area where the bacteria are labeled with a marker when the sample flow passes through it. Their role is to maintain the marker until the strip is used and to release the marker when there is recognition between the marker and a bacterium, when the sample flow passes. For example, the marking part and the marking zone are made of glass fibers, cellulose or polyesters.

For the detection of the capture of bacteria by bacteriophages or bacteriophage proteins, different types of markers can be used.

Thus, in a particular embodiment, the label can be any label capable of labeling a bacterium, a bacteriophage, a protein, in particular a bacteriophage protein, an antibody or an aptamer. Bacterial markers are well known to those skilled in the art.

The term "label" encompasses unconjugated labels and conjugated labels.

Within the meaning of the present application, an unconjugated marker is a free marker, that is to say that it is not bound to a ligand to mark the bacterium.

In the present application, the markers used can be colloidal molecules, in particular gold nanoparticles (AuNP). For example, they may be gold nanospheres or gold nanorods. The aggregation of gold nanoparticles leads to the appearance of a color, in particular a pink color, detectable in the visible, due to the absorbance wavelength of the gold nanoparticles. In the context of an ELISA-type device, the labeled element may be an antibody specific to the bacterium coupled with a substrate, for example horseradish peroxidase, which catalyzes the conversion of chromogenic substrates into colored compounds, or which produces light from chemiluminescent substrates; we can note ABTS, OPD, DAB (in), AEC (in) or even TMB (3,3',5,5'-Tetramethylbenzidine). In the present application, it is an antibody coupled with a peroxidase enzyme and the revelation of the color is made thanks to the addition of TMB.

Within the meaning of the present application, a conjugated label means that the label is bound to a ligand to label the bacterium. In particular, the conjugated marker is a marker conjugated to at least one specific ligand of the bacterium of interest. By “conjugated to at least one specific ligand”, it is meant that a label can be conjugated to a specific ligand or to several different specific ligands.

Unless otherwise stated, within the meaning of the present invention, said at least one ligand specific for the bacterium of interest can be at least one antibody directed against the bacterium of interest, at least one aptamer specific for the bacterium of interest, at least at least one bacteriophage specific for the bacterium of interest, at least one protein of at least one bacteriophage specific for the bacterium of interest, at least one bacteriophage specific for the bacterium of interest coupled to an antibody directed against the bacterium of interest, at least one protein of a bacteriophage specific for the bacterium of interest coupled to an antibody directed against the bacterium of interest. In particular, the bacterium of interest is S. aureus and these specific ligands read as ligands specific for S. aureus or directed against S. aureus . In particular, when the ligand is specific for S. aureus , said at least one bacteriophage specific for S. aureus is a bacteriophage CNCM I-5408, a bacteriophage CNCM I-5409 and/or a bacteriophage CNCM I-5410, and/or a bacteriophage CNCM I-5491, and/or a bacteriophage CNCM I-5492 and said at least protein of at least one bacteriophage, in particular an RBP, is at least protein of bacteriophage CNCM I-5408, at least protein of bacteriophage CNCM I-5409 and/or at least bacteriophage CNCM I-5410 protein at least bacteriophage CNCM I-5491 protein, at least bacteriophage CNCM I-5492 protein.

Thus, the label conjugated to a ligand specific for the bacterium of interest, in particular S. aureus , can be a label conjugated to an antibody directed against the bacterium of interest, a label conjugated to an aptamer specific to the bacterium of interest, a marker conjugated to a phage specific to the bacterium of interest, a marker conjugated to a protein of a phage specific to the bacterium of interest, a marker conjugated to a phage specific to the bacterium of interest coupled to a antibody directed against the bacterium of interest, a marker conjugated to a protein of a specific phage of the bacterium of interest coupled to an antibody directed against the bacterium of interest, a marker conjugated to an antibody directed against the bacterium of interest coupled to a phage specific to the bacterium of interest or a marker conjugated to a protein of a phage specific to the bacterium of interest coupled to an antibody directed against the bacterium of interest.

In a more particular embodiment, the present invention relates to a strip for detecting S. aureus in a sample, comprising in order at least:

• a contact part comprising a contact zone with the sample;
• a labeling part comprising a labeling zone which comprises a label conjugated to at least one ligand specific for S. aureus , in particular a gold nanoparticle conjugated to at least one ligand specific for S. aureus ;
• a detection part comprising a zone for detecting S. aureus which comprises at least one bacteriophage specific for S. aureus and/or a protein of at least one bacteriophage specific for S. aureus , in particular an RBP protein,
• an absorbent part.

In particular, the labeling zone comprises a label conjugated to an anti- S antibody . aureus or marker conjugated to an aptamer specific to S. aureus , more particularly a gold nanoparticle conjugated to an anti- S . aureus or a gold nanoparticle conjugated to an aptamer specific to S. aureus .

In particular, the labeling zone comprises a label conjugated to at least one bacteriophage specific for S. aureus . In particular, it comprises a marker conjugated to phage CNCM I-5408, a marker conjugated to phage CNCM I-5409, and/or a marker conjugated to phage CNCM I-5410, and/or a marker conjugated to phage CNCM I-5491 , and/or a marker conjugated to CNCM I-5492 phage. In particular, it comprises these three markers conjugated to phages. More particularly, said label conjugated to these phages is a gold nanoparticle.

In particular, the labeling zone comprises a label conjugated to at least one protein of at least one bacteriophage specific for S. aureus . In particular, said at least one protein is an RBP. In particular, it comprises a marker conjugated to at least one protein from phage CNCM I-5408, a marker conjugated to at least one protein from phage CNCM I-5409, and/or a marker conjugated to at least one protein from phage CNCM I -5410, and/or a marker conjugated to at least one protein from phage CNCM I-5491, and/or a marker conjugated to at least one protein from phage CNCM I-5492. In particular, it comprises these three markers conjugated to phage proteins. Even more particularly, said label conjugated to these phage proteins is a gold nanoparticle.

In particular, the labeling zone comprises a label conjugated to phage CNCM I-5408 coupled to an anti- S antibody . aureus , a CNCM I-5409 phage-conjugated marker coupled to an anti- S . aureus , a CNCM I-5410 phage-conjugated marker coupled to an anti- S . aureus , a CNCM I-5491 phage-conjugated marker coupled to an anti- S . aureus and/or a marker conjugated to the CNCM I-5492 phage coupled to an anti- S . aureus . In particular, it comprises a marker conjugated to a protein of the CNCM I-5408 phage coupled to an anti- S antibody . aureus , a CNVM I-5409 phage protein-conjugated marker coupled to an anti- S . aureus and/or a marker conjugated to a CNCM I-5410 phage protein coupled to an anti- S . aureus and/or a marker conjugated to a CNCM I-5491 phage protein coupled to an anti- S . aureus , and/or a marker conjugated to a CNCM I-5492 phage protein coupled to an anti- S . aureus . In particular, it is a marker conjugated to an anti- S antibody . aureus coupled to phage CNCM I-5408, a marker conjugated to an anti- S . aureus coupled to phage CNCM I-5409 and/or a marker conjugated to an anti- S antibody . aureus coupled to phage CNCM I-5410, and/or a marker conjugated to an anti- S antibody . aureus coupled to the CNCM I-5491 phage, and/or a marker conjugated to an anti- S antibody . aureus coupled to CNCM I-5492 phage. In particular, it is a marker conjugated to an anti- S antibody . aureus coupled to a protein from phage CNCM I-5408, a marker conjugated to an anti- S . aureus coupled to a CNCM I-5409 phage protein and/or a label conjugated to an anti- S antibody . aureus coupled to a protein from phage CNCM I-5410, and/or a label conjugated to an anti- S antibody . aureus coupled to a protein from phage CNCM I-5491, and/or a marker conjugated to an anti- S antibody . aureus coupled to a protein from phage CNCM I-5492.

In particular, the detection zone comprises a CNCM I-5408 phage, a CNCM I-5409 phage and/or a CNCM I-5410 phage, and/or a CNCM I-5491 phage and/or a CNCM I-5492 phage. In particular, the detection zone comprises a CNCM I-5408 phage, a CNCM I-5409 phage and a CNCM I-5410 phage. In particular, the detection zone comprises CNCM I-5408 phage, CNCM I-5409 phage, CNCM I-5410 phage, In particular, the detection zone comprises CNCM I-5408 phage, CNCM I-5409 phage , a CNCM I-5491 phage and a CNCM I-5492 phage.

In particular, the detection zone comprises at least one protein from phage CNCM I-5408, at least one protein from phage CNCM I-5409, and/or at least one protein from phage CNCM I-5410, and/or at least one protein from phage CNCM I-5491, and/or at least one protein from phage CNCM I-5492. In particular, the detection zone comprises at least one protein from phage CNCM I-5408, at least one protein from phage CNCM I-5409 and at least one protein from phage CNCM I-5410. In particular, the detection zone comprises at least one protein from phage CNCM I-5408, at least one protein from phage CNCM I-5409 and at least one protein from phage CNCM I-5410, and at least one protein from phage CNCM I -5491 and at least one protein from phage CNCM I-5492.

In another embodiment, said strip (for lateral flow chromatography) comprises in order at least:

• a contact part comprising a contact zone with the sample;
• a labeling part comprising a labeling zone which comprises a label conjugated to at least one ligand specific for the bacterium of interest chosen from at least one bacteriophage specific for the bacterium of interest, at least one protein of at least one bacteriophage specific for the bacterium of interest, at least one bacteriophage specific for the bacterium of interest coupled to an antibody directed against the bacterium of interest, at least one protein of a bacteriophage specific for the bacterium of interest coupled to an antibody directed against the bacterium of interest,
• a detection part comprising a zone for detecting the bacterium of interest which comprises at least one ligand specific for the bacterium of interest,
• an absorbent part.

In particular, said at least one ligand specific for the bacterium of interest included in the detection zone is at least one antibody directed against the bacterium of interest, at least one aptamer specific for the bacterium of interest, at least one bacteriophage specific for the bacterium of interest, at least one protein of at least one bacteriophage specific for the bacterium of interest, at least one bacteriophage specific for the bacterium of interest coupled to an antibody directed against the bacterium of interest, at the at least one bacteriophage protein specific for the bacterium of interest coupled to an antibody directed against the bacterium of interest.

In a more particular embodiment, the present invention relates to a strip for detecting S. aureus in a sample, comprising in order at least:

• a contact part comprising a contact zone with the sample;
• a labeling part comprising a labeling zone which comprises a label conjugated to at least one ligand specific for S. aureus chosen from: at least one bacteriophage specific for S. aureus , at least one protein of at least one bacteriophage specific for S. aureus, at least one bacteriophage specific for S. aureus coupled to an antibody directed against S. aureus , at least one protein of a bacteriophage specific for S. aureus coupled to an antibody directed against S. aureus ;
• a detection part comprising an S. aureus detection zone which comprises at least one ligand specific for S. aureus ;
• an absorbent part.

In particular, the labeling zone comprises a label conjugated to at least one bacteriophage specific for S. aureus . In particular, it comprises a marker conjugated to phage CNCM I-5408, a marker conjugated to phage CNCM I-5409, and/or a marker conjugated to phage CNCM I-5410, and/or a marker conjugated to phage CNCM I-5491 , and/or a marker conjugated to CNCM I-5492 phage. More particularly, it comprises a marker conjugated to phage CNCM I-5408, a marker conjugated to phage CNCM I-5409, a marker conjugated to phage CNCM I-5410, a marker conjugated to phage CNCM I-5491 and a marker conjugated to phage CNCM I-5492. More particularly, it comprises a marker conjugated to phage CNCM I-5408, a marker conjugated to phage CNCM I-5409 and a marker conjugated to phage CNCM I-5410. Even more particularly, said label conjugated to these phages is a gold nanoparticle.

In particular, the labeling zone comprises a label conjugated to at least one protein of at least one bacteriophage specific for S. aureus. In particular, said at least one protein is an RBP. In particular, it comprises a marker conjugated to at least one protein from phage CNCM I-5408, a marker conjugated to at least one protein from phage CNCM I-5409, and/or a marker conjugated to at least one protein from phage CNCM I -5410, and/or a marker conjugated to at least one protein from phage CNCM I-5491, and/or a marker conjugated to at least one protein from phage CNCM I-5492. More particularly, it comprises a marker conjugated to at least one protein from phage CNCM I-5408, a marker conjugated to at least one protein from phage CNCM I-5409 and a marker conjugated to at least one protein from phage CNCM I-5410. More particularly, it comprises a marker conjugated to at least one protein from phage CNCM I-5408, a marker conjugated to at least one protein from phage CNCM I-5409, a marker conjugated to at least one protein from phage CNCM I-5410, a marker conjugated to at least one protein from phage CNCM I-5491 and a marker conjugated to at least one protein from phage CNCM I-5492. Even more particularly, said marker conjugated to these phage proteins is a gold nanoparticle.

In particular, the detection zone comprises an anti- S aureus antibody or an aptamer specific for S. aureus. In particular, the detection zone comprises at least one bacteriophage specific for the bacterium of interest, more particularly a bacteriophage CNCM I-5408, a bacteriophage CNCM I-5409 and/or a bacteriophage CNCM I-5410, and/or a bacteriophage CNCM I-5491, and/or a CNCM I-5492 bacteriophage. More particularly, it comprises these three bacteriophages. Even more particularly, More particularly, it comprises these five bacteriophages.

In particular, the detection zone comprises at least one protein from at least one bacteriophage specific for S. aureus , in particular an RBP. More particularly, it comprises at least bacteriophage CNCM I-5408 protein, at least bacteriophage CNCM I-5409 protein and/or at least bacteriophage CNCM I-5410 protein, and/or at least bacteriophage CNCM I-5491 protein, and/or at least bacteriophage CNCM I-5492 protein. Even more particularly, it comprises at least bacteriophage CNCM I-5408 protein, at least bacteriophage CNCM I-5409 protein and at least bacteriophage CNCM I-5410 protein. Even more particularly, it comprises at least bacteriophage CNCM I-5408 protein, at least bacteriophage CNCM I-5409 protein, at least bacteriophage CNCM I-5410 protein, at least bacteriophage CNCM I-5491 protein and at least bacteriophage protein CNCM I-5492 bacteriophage.

In particular, the detection zone comprises at least one bacteriophage specific for S. aureus coupled to an antibody directed against S. aureus and/or at least one protein of a bacteriophage specific for S. aureus coupled to an antibody directed against S. aureus , in particular in which said bacteriophage is chosen from bacteriophages CNCM I- 5408, CNCM I-5409, CNCM I-5410, CNCM I-5410, CNCM I-5491 and CNCM I-5492 and said protein is chosen from the proteins of these phages.

In a particular embodiment, when the strip comprises at least four parts, one of the ends of the contact part is superimposed with one of the ends of the part downstream of the latter to form a first zone of superposition ; one of the ends of the marking part is superimposed with one of the ends of the part upstream of the latter to form a second overlapping zone and the other end of the marking part is superimposed with one ends of the part downstream thereof to form a third superposition zone; one of the ends of the detection part is superimposed with one of the ends of the upstream part thereof to form a fourth superposition zone and the other end of the detection part is superimposed with one ends of the part downstream thereof to form a fifth overlapping zone; and one of the ends of the absorbent part is overlapped with one of the ends of the part downstream thereof to form a sixth zone of overlap. In particular, the first, the second, the third, the fourth, the fifth and the sixth overlapping zone are separated from each other. In particular, the contact zone is distinct from the first overlapping zone. In particular, the marking zone is distinct from the second and from the third overlapping zone. In particular, the detection zone is distinct from the fourth and from the fifth overlapping zone.

In a particular embodiment, one of the ends of the marking part is superimposed on one of the ends of the contact part and the other end of the marking part is superimposed on one of the ends of the detection part; the other end of the detection part being superimposed with one of the ends of the absorbing part.

In a particular embodiment, one of the ends of the marking part is superimposed on one of the ends of the contact part to form a first superposition zone, and the other end of the marking part is superimposed at one of the ends of the detection part to form a second overlapping zone; the other end of the detection part being superposed with one of the ends of the absorbing part to form a third superposition zone. In particular, the first, the second and the third overlap zone are separated from each other. In particular, the contact zone is distinct from the first overlapping zone. In particular, the marking area is distinct from the first and from the second overlapping area. In particular, the detection zone is distinct from the second and from the third superposition zone.

In a particular embodiment, the detection zone is a line transverse to the strip, thus forming a detection line or test line.

In a particular embodiment, the detection zone is distributed and is limited by a line transverse to the strip.

Optionally, the detection part can also comprise a control zone as described previously.

ELISA test

The principle of ELISA consists in trapping, between a "capture antibody" and a "detection antibody", the analytes (antigens, bacteria, viruses, etc.). The specificity of the detection system of the present invention compared to the conventional ELISA system is the replacement of one or both antibodies by phages or phage proteins.

In the context of implementing the present invention in the form of an ELISA test, the detection and/or quantification of bacteria in a sample is carried out by replacing one of the two antibodies or the two antibodies of the conventional ELISA test by phages or phage proteins.

In a particular embodiment of the invention, it is possible to replace the antibody coupled to a marker with phage proteins or phages coupled directly or indirectly to a marker (gold or other probe).

It is possible to use an antibody for the revelation and/or detection of the presence of bacteria in the sample.

It is possible to use several phages or phage proteins or to combine them with antibodies in order to capture several bacterial strains (detection spectrum).

In this system instead of using the antibody coupled to HRP, it is possible to use the antibody coupled to a colloidal molecule such as gold or another label molecule in order to measure the absorbance directly without the need for the stage of revelation by TMB.

According to one embodiment, phages or phage proteins are used for the capture of bacteria (analytes) and an antibody is used for detection ( ). In this particular implementation, the phages or phage proteins are fixed on the support using a solution comprising a cation and a protein (in particular a solution of MgSO4 and BSA). In another implementation of the invention, the phage proteins, in particular the RBPs, are fixed to the support using a solution comprising a cation or a protein.

It is also possible to replace the antibody coupled to a label with phage proteins or phages coupled directly or indirectly to a label (gold or other probe) for the detection of bacteria (Figure 7 B). In another implementation of the invention phage proteins, in particular RBPs, are used for the detection of bacteria and are used in a solution comprising a cation and a protein or in a solution comprising a cation or a protein ( in particular a solution of MgSO4 and BSA).

Thus, alternative embodiments of the present invention include the use of phages or phage proteins for the capture and detection of bacteria or the use of an antibody for the capture of bacteria and the use of phages or proteins of phages for detection ( ). In another implementation of the invention phage proteins, in particular RBPs, are used for the capture and detection of bacteria and are used in a solution comprising a cation and a protein or in a solution comprising a cation or a protein (especially a solution of MgSO4 and BSA)

It is also possible to use several phages or phage proteins or to combine them with the antibodies in order to capture several bacterial strains (detection spectrum).

BRIEF DESCRIPTION OF FIGURES

is a schematic representation of an embodiment M1 in which A is a marker for the target bacterium, examples: antibody specific to the bacterium coupled to a label or phage/phage protein coupled to a label or a marker which stains the bacterium and the solid oval is a bacterium. The marker is deposited on the marking part. The phages/phage proteins are deposited in a line at the cellulose membrane.

is a schematic representation of an embodiment M2 in which A is a marker of the target bacterium, examples: antibody specific to the bacterium coupled to a label or phage/phage protein coupled to a label or a marker which stains the bacterium The marker is deposited on the marking part. Phages/phage proteins are deposited all along the bottom of the cellulose membrane.

is a schematic representation of an embodiment M3 identical to M1 but in which the bacteria are stained in the solution by a marker in which A is a marker for the target bacterium, examples: antibody specific to the bacterium coupled to a label or phage/phage protein coupled to a tag or marker that stains the bacteria in solution. The phages/phage proteins are deposited in a line at the cellulose membrane.

is a schematic representation of an embodiment M4 identical to M2 but in which the bacteria are stained in the solution by a marker and in which A is a marker of the target bacterium, for example: an antibody specific to the bacterium coupled to a label or phage/phage protein coupled to a label or a marker which stains the bacterium. Phages/phage proteins are deposited all along the bottom of the cellulose membrane.

is a schematic representation of an embodiment M5 identical to M1 but the phages / phage proteins and the bacteria marker are deposited on the labeling part (PAD conjugate) and in which A is a marker of the target bacterium, for example an antibody specific to the bacterium coupled to a label or phage/phage protein coupled to a label or a marker which stains the bacterium. The phages/phage proteins are also deposited on the labeling part (PAD conjugate).

is a schematic representation of an embodiment M6 identical to M5 but the phages/phage proteins on the tag portion (PAD Conjugate) and not the tag. The bacteria are stained in the solution and wherein A is a marker of the target bacterium, for example: an antibody specific to the bacterium coupled to a label or phage/phage protein coupled to a label or a marker which stains the bacterium in the solution.

represents an embodiment of the invention in an ELISA type device.

represents an embodiment of the invention in an ELISA type device.

represents an embodiment of the invention in an ELISA type device.

is a photograph showing the detection of S. aureus on a strip to which bacteriophage proteins are attached. The effect of solution A alone is tested (solution containing MgSO4), solution B (BSA) or the combination of solutions A and B.

is a photograph showing the detection of S. aureus on a strip to which bacteriophage proteins are attached. Different concentrations of phages are tested as well as the absence of bacteria in the sample

is a photograph showing a microtiter plate showing the presence of S. aureus.

is a graph showing the detection threshold of S. aureus on a strip to which are attached bacteriophage proteins.

is a histogram showing the link between the measured optical density (OD) and the concentration of bacteria expressed in CFU/mL, a control without bacteria is present (TM (without bacteria)).

is a photo showing that the expression of RBP proteins of phages 2, 3 and 7 in BL21 bacteria was verified on gel ( ).

is a histogram which shows that the RBPs proteins of phages 2, 3 and 7 have an affinity for the species S. aureus, ie, no affinity for the bacteria S. epidermis or S. algenitis

EXAMPLES

The present invention will be better understood on reading the following examples which illustrate the invention without limitation.

Example 1: Bacteriophage activity

1.1. Determination of the activity of a bacteriophage on a bacterial strain

To identify whether a bacteriophage is specific to a bacterium, the spot method is used. On a Petri dish on which is spread a culture of bacteria in the exponential phase, at least one spot comprising the bacteriophage is deposited. If a lysis patch is observed around the spot, this means that the bacteriophage is active for this bacterium, in other words that the bacteriophage is specific for this bacterium.

1.2. Bacteriophages used

Five phages were tested in this application:

Bacteriophage strains

The present invention relates to the bacteriophage strain deposited at the National Collection of Microorganism Cultures (CNCM) under the number CNCM I-5408.

The present invention also relates to the bacteriophage strain deposited at the CNCM under the number CNCM I-5409.

The present invention also relates to the bacteriophage strain deposited at the CNCM under the number CNCM I-5410.

The present invention also relates to the bacteriophage strain deposited at the CNCM under the number CNCM I-5491.

The present invention also relates to the bacteriophage strain deposited at the CNCM under the number CNCM I-5492.

1. Determination of the activity spectrum of isolated phages

According to the digestion profiles, nine phages were finally selected to test their activity on different strains of S. aureus isolated from the breeding samples.

For phages 2 to 7, the activity of the isolated phages is compared with that of the reference phage which is phage K (phage 1).

Results

The spectrum of activity was tested on the 30 strains of S. aureus (Table 1) or on 25 bacterial strains including 19 strains of S. Aureus and 6 non- S. Aureus strains (Table 2) by the spot method. The intensity of lysis of these phages on each of the bacterial strains is detailed in Table 1 and Table 2. Table 1 therefore represents the spectrum of activity of the phages isolated on the strains of bovine S. aureus and Table 2 represents the spectrum of activity of the phages isolated on different bacterial strains. The lysis obtained by the spot method is noted from (-)=no activity to (++++)=very active.

Bacteria/phages 1 2 3 4 5 6 7 Cocktail of 3 phages (2, 3, and 7) 1 76.25 +++++ ++++ - ++ + - +++ +++ 2 79 +++++ +++++ ++++ +++++ ++++ +++++ ++++ ++++ 3 80 +++++ +++++ ++++ ++++ +++ ++++ ++ 4 81 +++++ +++++ ++++ +++++ +++ ++C ++C ++++ 5 Q7 +++++ ++++ ++ + ++ - +++ ++++ 6 Q12.b +++++ ++++ +++ + +++ - -(+) +++ 7 Q14.b - ++ - + + - +++ +++ 8 C24.b +++++ ++++ ++ - ++ - ++ ++++ 9 F8.a1 ++++ ++++ +++ - +++ - ++ ++++ 10 F2.b +++++ +++ +++ - ++ - ++ ++++ 11 F5.a +++++ ++++ +++ - +++ - + +++ 12 F8c +++++ +++++ ++ ++ - - +++ ++++ 13 L1.a +++++ +++++ - ++ - - +++ +++ 14 L1.b +++++ +++ - ++ - - +++ +++ 15 L3.b +++++ ++++ - + - - +++ +++ 16 L3c +++++ ++++ + + - - +++ +++ 17 L5.a +++++ ++++ - + - - +++ +++ 18 L5b +++++ ++++ - +++ - - ++++ ++++ 19 G6.b - - + + +++ - ++ +++ 20 U22b +++++ ++++ ++++ +++ - +++ +++ ++++ 21 Z12c2 ++++ ++++ +++ C - - ++C +++C ++++ 22 U7b2 +++++ ++++ +++ C +C - + -(+) ++++ 23 U20b +++++ ++++ ++++ +++ ++ ++C ++++ ++++ 24 U18b +++++ +++++ +++ C +C - ++C ++C ++++ 25 U1b2 +++++ +++++ ++++ + - ++ ++ ++++ 26 U4a +++++ +++++ ++++ + - ++ ++ ++++ 27 U11a +++++ +++++ +++ C +C +C ++C +++ C ++++ 28 U6d +++++ +++++ ++++ + - +++ +++ ++++ 29 U5a +++++ +++++ +++ C +++ C - + ++ ++++ 30 3 +++++ +++++ - ++ - ++ +++ ++++ 28/30 29/30 22/30 25/30 14/30 14/30 28/30 30/30 93% 97% ¨73% 83% 47% 47% 93% 100%

Phage/
Strain Bacterial strains/Phages Page 6 Phage 3 phage 8 Page 9 Phage10 Page 2 phage 5 phage 7 Mix 1 (2/3/7) Mix 2 (2/9/10) 79 1 S. aureus ++++ ++++ ++++ +++++ +++++ +++++ - ++++ ++++ ++++ 81 2 S. aureus +++ +++ +++++ +++++ +++++ +++++ ++ ++ ++++ ++++ C24.b 3 S. aureus - +++ ++ ++ +++ +++++ + ++ ++++ ++++ F1.a 4 S. aureus - +++ ++ ++ +++ +++++ ++ - ++++ ++++ L5.a 5 S. aureus - - - - - ++++ +++ +++++ ++++ ++++ Q12.b 6 S. aureus - ++ ++ ++ +++ +++++ + +++++ ++++ ++++ Q14.b 7 S. aureus - - - - - - + ++ + ++ U5a.a 8 S. aureus ++ +++ +++++ +++++ ++++ +++++ ++ ++++ ++++ ++++ F2.a 9 S. aureus - +++ ++ ++ +++ +++++ + - +++ ++++ L1.a 10 S. aureus ++ ++ - ++ ++ +++++ ++ ++++ ++++ ++++ G6.b 11 S. aureus - - - - - +++ ++ ++ ++ +++ To 12 S. aureus - ++++ ++ ++ +++ +++++ + - +++ ++++ 3 13 S. aureus ++ - - - - +++++ - +++ ++++ ++++ R8c2 14 S. aureus - ++ ++ ++ +++ +++++ + - ++++ ++++ 1Da2 15 S. aureus - - - + ++ - + - + ++ Z12c2b 16 S. aureus ++ ++ ++++ +++++ ++++ +++++ + +++++ +++ +++ 5S.d 17 S. aureus - - ++ +++ ++ ++++ - ++ +++ +++ 9L.a 18 S. aureus - +++ ++ ++ ++++ +++++ ++ ++ +++ ++++ Mockingbird 19 S. aureus - - ++ ++ +++ +++++ ++ - ++++ ++++ 31.50% 63.00% 68.40% 79% 79% 89% 68.40% 100% 100%

The assessment of the lysis intensity is assessed from 0 (-) to ++++). The notation "C" means that a few colonies are observed in the lysis range.

These results show that:

• Phage K (ATCC 19685-B1) (phage 1) infects 28 strains out of the 30 strains tested, ie 93% (Table 1).
• The bacteriophage deposited at the CNCM under the number CNCM I-5408 (phage 2) infects 29 strains out of the 30 strains tested, ie 97% (Table 1);
• The bacteriophage deposited at the CNCM under the number CNCM I-5409 (phage 3) infects 22 strains out of the 30 strains tested, ie 73% (Table 1);
• Bacteriophage 4 infects 25 strains out of the 30 strains tested, i.e. 83% (Table 1);
• Bacteriophage 5 infects 14 strains out of the 30 strains tested, ie 47% (Table 1);
• Bacteriophage 6 infects 14 strains out of the 30 strains tested, i.e. 47% (Table 1);
• The bacteriophage deposited at the CNCM under the number CNCM I-5410 (phage 7) infects 28 strains out of the 30 strains tested, i.e. 93% (Table 1);
• The cocktail of three phages (2, 3 and 7) infects 30 strains out of the 30 strains tested, i.e. 100% (Table 1);
• Bacteriophage 8 infects 14 strains out of 19 S. aureus strains, i.e. 68.4% (Table 2)
• The bacteriophage deposited at the CNCM under the number CNCM I-5491 (phage 9) infects 15 strains out of 19 S. aureus strains, i.e. 79% (Table 2);
• The bacteriophage deposited at the CNCM under the number CNCM I-5492 (phage 10) infects 15 strains out of 19 S. aureus strains, i.e. 79% (Table 2);
• The cocktail of three phages (2, 9 and 10) infects 19 strains out of 19 S. aureus strains, ie 100% of the S. aureus strains tested (Table 2).

The activity spectrum of phage 2 (CNCM I-5408) is the broadest and comparable to phage K (reference phage). However, phage K cannot infect all strains tested. On the other hand, certain mixtures of bacteriophages according to the invention make it possible to infect more strains of S. aureus than phage K, or even 100% of the strains of S. aureus tested.

In particular, it was shown that the mixture of phages n°CNCM I-5408 (phage 2), n°CNCM I-5409 (phage 3), and n°CNCM I-5410 (phage 7) made it possible to infect the all of the 30 strains, i.e. 100% of the strains tested (Table 1).

It was also shown that the mixture of phages n°CNCM I-5408 (phage 2), n°CNCM I-5491 (phage 9), and n°CNCM I-5492 (phage 10) made it possible to infect all the 19 S Aureus strains, i.e. 100% of the S. Aureus strains tested (Table 2).

The phages according to the invention have a broad spectrum of activity on the infection of strains of S. aureus and were also selected for their rate of elimination of S. aureus (after 3 h of incubation at 37° C. in BHI).

Example 2: Selection of Bacteriophage Proteins

2.1. Choice of bacteriophage proteins

The adsorption of bacteriophages in the host bacterium is carried out thanks to the specific recognition on the bacterial surface, of the phage binding proteins (Receptor Binding Protein or RBP), located at the base (“baseplate”) of the tail of the phages ( ).

In order to determine the binding specificity of different types of phage proteins to S. aureus , the following proteins were selected and synthesized:

• a phage 2 RBP,
• a phage 3 RBP,
• a phage 7 RBP.

For this, phages 2, 3 and 7 were sequenced. The search for RBPs was carried out using complete annotations of the genomes of phages 2, 3 and 7, targeting the sequences of the “flat base” region. The alignment of the "flat base" sequences of each phage was carried out using the BlastX technique of the ORFs (open reading frame) of the phages.

The RBPs proteins were found at the level of the tail gene cluster around 55,000 bp to 84,000 bp and more precisely at the “flat base” part.

The RBPs proteins of phages 2, 3 and 7 were then produced in the bacterium BL21 by the cloning technique. For this, the genes corresponding to the phage proteins were cloned into a bacterial expression vector allowing the synthesis of proteins in fusion with the enzyme Glutathione-S-Transferase (GST) by the host bacterium BL21.

Assessing phage protein binding specificity to S. aureus

After purification, the proteins were separately conjugated with magnetic beads so that the surface of these beads was by one of the RBP proteins.

To assess the binding specificity of each of these proteins to the bacterial species S. aureus, the magnetic beads conjugated to the proteins to be tested were separately added to a mixture of bacteria comprising different species of Staphylococci (Staphylococcus aureus, Staphylococcus epidermidis and Staphylococcus algnetis ).

If there is a specific interaction (affinity) between the tested protein and one or more bacterial species in the mixture, the protein will capture the bacteria(s).

The beads are then separated from the liquid phase using a magnetic support, and washed several times with a buffer solution in order to eliminate non-specific interactions.

The beads are then recovered and spread in a Petri dish on a specific agar (Baird Parker). After incubation, the bacterial species fixed by the proteins are visually identified according to the morphology and the color of the colonies developing on agar. The number of bacteria retained is also determined by counting the colonies on the agar. The affinity or the interaction of the protein tested with the target bacterium ( S. aureus ) is evaluated by the number of bacteria retained. The higher the number of target bacteria on the agar, the greater the affinity of the protein for the target bacteria.

2.3. Results

The expression of RBP proteins of phages 2, 3 and 7 in BL21 bacteria was verified on gel ( ). The respective sizes of the RBPs of phages 2, 3 and 7 are 78.1 kDa, 100.8 kDa and 79.5 kDa.

The binding specificity of these proteins to the bacterial species S. aureus was then studied. The results showed that the RBP gp68 protein of phage 2 has an affinity for the bacterium Staphylococcus aureus and only for this species of Staphylococcus. Moreover, the results in show that the RBPs proteins of phages 2, 3 and 7 have an affinity for the species S. aureus.

Example 3: different embodiments

According to one embodiment, the strip for detecting a bacterium, in particular S. aureus , by a strip, is composed of 4 parts, with in order:

• a contact part (sample pad);
• a marking part (conjugate pad);
• a detection part (test pad);
• an absorbent part (absorption pad); designed to absorb excess reagents or solutions; wherein one of the ends of the marking part is overlapped with one of the ends of the contact part, the other end of the marking part is overlapped with one of the ends of the detection part, and the other end of the detection part is superimposed with one of the ends of the absorbent part, to respectively form a first, a second and a third superposition zone, said superposition zones being separated from each other.

The contact part (sample pad) comprises a contact zone intended to be brought into contact with the sample. In particular, this contact zone is distinct from the first superposition zone. In particular, the contact part and the contact zone have the same composition. They are composed of a capillary matrix intended to promote and evenly distribute the sample for its migration by capillarity. Indeed, when the contact zone is brought into contact with the sample, a migration flow of the sample by capillarity, from the contact part to the absorbent part, is established.

The labeling part (Conjugate pad) includes a labeling area where a ligand conjugated to a label and capable of binding specifically to the bacterium of interest is deposited. In particular, the marker is a gold nanoparticle (AuNP). In particular, the ligand is an antibody directed against the bacterium of interest. In particular, the marking zone is on the marking part, between the first and the second overlapping zone.

When the sample fluid passes through the labeling area, the labeled ligand binds to the bacterium, forming a bacterium/labeled ligand complex. Said complex is then entrained by the migration flow through the labeling part, then through the detection part.

The detection part comprises a detection zone, where a phage specific to the bacterium of interest or a protein of a phage specific to the bacterium of interest is immobilized. In particular, the detection zone is on the detection part, between the second and the third overlapping zone.

In a particular embodiment, the detection zone is a line transverse to the strip, when the liquid is directed towards the test zone, the bacteria stained by a label are picked up by the phages / phage proteins resulting in a coloring zone related to bacteria blockage at the test line.

In particular, the detection part and the detection zone have the same composition. They are composed of a membrane, more specifically a nitrocellulose membrane.

When passing the test line, the bacteria/labeled ligand complexes are immobilized by the test line.

The presence of the analyte is then revealed thanks to the marker, by the appearance of a visual signal on the strip. In particular, when the marker is a gold nanoparticle, the accumulation of AuNPs on the migration zone up to the test line leads to the appearance of a visual signal, in particular of pink color.

There illustrates one embodiment of a strip for detecting S. aureus in a sample, comprising in order the following four parts:

• a contact part comprising a contact zone with the sample to be tested;
• a labeling part comprising a labeling zone where an antibody directed against S. aureus labeled with a gold nanoparticle (AuNP) is deposited;
• a detection part comprising an S. aureus detection zone, where a bacteriophage specific for S. aureus or a bacteriophage protein specific for S. aureus is immobilized,
• an absorbent part.

In particular, the first ligand is labeled with a gold nanoparticle. The visual signal will be the appearance of a pink color along the strip.

Example 4: strip system

The bacteria detection strip system according to the invention is a strip that phages and phage proteins can be used. In the strip system according to the invention, the fixing of the phages or the phage proteins using the specific solution which causes a response of the presence of the bacterium by blocking the migration due to the phages or the protein fixed on the membrane.

Binding of phage proteins can capture bacteria. The presence of the bacterium is reflected by the blocking of migration ( ).

In the context of this example, compound A is BSA. It is used at a concentration of 2 mg/mL, compound B is MgSO ₄ . It is used at a concentration of 50 mM.

There shows an example where phage proteins have been immobilized in a solution containing A without B (left); or a solution containing B without A (middle) and in a solution containing both A and B (right). Strips were dipped in raw milk without bacteria (left) or with bacteria (right) from each condition. When the phage proteins prepared in the solution containing both Compound A and B, the bacteria were picked up at the area where the proteins were immobilized.

The strip system detected bacteria at 10 ³ CFU/mL in a sample of raw milk ( ). The signal is different when the bacterium is present in the medium and the absence of the bacterium.

Conclusion :

The strip system according to the invention is based on phage or phage protein binding using the specific phage or phage protein binding formulation; the signal reflecting the presence of the bacterium is not represented by a colored line as in the classic system but by a blocking of migration. This type of signal is never observed in the literature or on the market.

The detection sensitivity makes the essential difference between the conventional system and the system of the invention.

Example 5: ELISA embodiment

The principle of ELISA consists in trapping, between a "capture antibody" and a "detection antibody", the analytes (antigens, bacteria, viruses, etc.). The specificity of the detection system of the present invention compared to the conventional ELISA system is the replacement of one or both antibodies by phages or phage proteins.

The phages or the phage proteins are prepared in a coating buffer containing MgSO ₄ (50 mM) and BSA (2 mg/mL). The solution is distributed in the wells of a microtiter plate. The plate is then covered and placed in an enclosure at 4° C. overnight.

The following day each well several times (at least 3 times) with a rinsing buffer (PBS buffer optionally contains Tween 0.5%).

Optionally, non-specific interactions are then blocked with a saturation buffer.

The sample potentially containing bacteria is distributed at a rate of 100 µl of sample in each well. A one-hour incubation at room temperature follows. The sample is removed and the wells are rinsed at least 3 times with Rinse Buffer (PBS Buffer optionally contains 0.5% Tween).

The antibody to S. aureus , Staphylococcus aureus Rabbit Polyclonal Antibody supplied by Thermofisher (Ref. 11578351), conjugated to HRP is added. Incubation at least 30 min at T° from 4°C to 37°C.

The antibody solution is removed and the wells are rinsed at least 3 times with the rinsing buffer (PBS buffer which optionally contains Tween 0.5%).

The presence of bacteria is revealed by adding TMB (ready to use).

The reaction is stopped by adding 100 µL of 1 M sulfuric acid.

A reading of the optical density is taken (Figure 10). The fixation of the phages or phage proteins was carried out in the solution containing both the two compounds A and B (specific formulation).

The main stages of the test:

INTERPRETATION OF RESULTS/ANALYSIS

The plate reader gives corresponding optical densities (OD) to each well, to be extracted into a spreadsheet for processing. The determination of the concentrations is done according to the following steps:

• Subtract from each OD the OD corresponding to 0 (buffer OD)
• Determine the equation of the range (which is generally linear, with the concentrations of range X and the corresponding DOs in Y): DO=A*[C] + B
• From this range equation (Figure 11) determine the concentrations by introducing the OD values obtained for the samples. Reduce these concentrations to the real values according to the dilution of the sample operated.

Detection of S. aureus using S. aureus phage protein for capture (attached to plate wells) and anti-S. aureus coupled to HRP for revelation by TMB. The system allowed detection of S. aureus at a threshold between 102 to 103 bacteria/mL (Figure 10).

References to deposited biological material

In this application, reference is made to the following bacteriophage strains, filed at the CNCM on March 21, 2019 by Vetophage SAS:

• phage 2, identification reference with the CNCM “Vetophage – phi 2”, whose registration number is “CNCM I-5408”;
• phage 3, identification reference with the CNCM “Vetophage – phi 3”, whose registration number is “CNCM I-5409”.

In this application, reference is made to the following strains of bacteriophages, submitted to the CNCM on February 12, 2020 by Vetophage SAS:

• phage 9, identification reference with the CNCM “Vetophage – phi 9”, whose registration number is “CNCM I-5491”;
• phage 10, identification reference with the CNCM “Vetophage – phi 10”, whose registration number is “CNCM I-5492”. 

Claims (15)

Use of a solution comprising a cation and a protein, for the detection or quantification of a bacterium by a bacteriophage or a bacteriophage protein. Use of a solution comprising a cation and a protein, for the detection or quantification of a bacterium by a bacteriophage or a bacteriophage protein according to claim 1, wherein said bacteriophage protein is a receptor binding protein (RBP ). Use according to claim 1 or 2 wherein the protein is a milk protein, albumin, bovine serum albumin (BSA), serum, chicken serum or an antibody. Use according to one of Claims 1 to 3 , in which the cation is chosen from divalent cations, including Mg2 ⁺ or Ca ²⁺ or else the cations Na ⁺ , K ⁺ , Fe ²⁺ , Fe ³⁺ , K ⁺ , Zn ⁺ , Au ³⁺ , Cu ⁺ , carbocation, Mn ⁺ , Ag ⁺ , or the salts MgSO4, Na2SO4, MgCl2, CaCl2, CaSO4, K2SO4 or KCl. Use according to any of the claims1To4in which

• the cation concentration, in particular MgSO4, in the solution is between 5 mM and 300 mM inclusive, and
• the protein concentration, in particular BSA, in the solution is between 0.1 mg/mL to 5 mg/mL inclusively.

Use according to any one of claims 1 to 5 , wherein said bacterium is Staphylococcus aureus and said bacteriophage is specific for Staphylococcus aureus . Use according to Claim 6 , characterized in that the said bacteriophage is chosen from bacteriophages specific for S. aureus as deposited at the CNCM under the numbers CNCM I-5408, CNCM I-5409, CNCM I-5410, CNCM I-5491 and CNCM I-5492, or is a mixture of these bacteriophages Use according to any one of claims 1 to 7 , wherein the bacteriophage protein is a receptor binding protein (RBP), specific for Staphylococcus aureus . Use according to claim 8 wherein the receptor binding protein (RBP) is selected from bacteriophage CNCM I-5408 RBP, bacteriophage CNCM I-5409 RBP, bacteriophage CNCM I-5410 RBP or a mixture of those -this. Use according to claim 9 , in which the receptor-binding protein (RBP) is chosen from an RBP of sequence SEQ ID NO: 1, an RBP of sequence SEQ ID NO: 2, an RBP of sequence SEQ ID NO: 3, or a mixture of these A method of preparing a device comprising a solid support for the detection or quantification of a bacterium by a bacteriophage or a bacteriophage protein, said method comprising:

• a step a) of coupling a bacteriophage or of a combination of bacteriophages or of a protein of bacteriophages or of a combination of proteins of bacteriophages on the solid support, the said coupling step consisting in bringing the support into contact with a bacteriophage or a combination of bacteriophages or a protein of bacteriophages or a combination of proteins of bacteriophages in the presence of a solution comprising a cation and a protein, the bacteriophage protein preferably being an RBP,
• optionally a step b) of eliminating the bacteriophage(s) or the bacteriophage protein(s) not bound to the solid support, and
• optionally a step c) of rinsing, which can be repeated at least once.

Device obtained according to the preparation process according to claim 11 , said device being a microtiter plate or a strip. A device according to claim 12 comprising a solid support having attached thereto a receptor binding protein (RBP), the support being pre-coated with a protein. Use of the device according to any one of claims 12 or 13 for the detection or quantification of one or more bacteria present in a sample. Method for the detection or quantification of one or more bacteria present in a sample with a device of the strip or ELISA type, comprising the following steps

1. bringing the sample likely to contain bacteria into contact with a device according to any one of claims 12 or 13 ,
2. optionally elimination of bacteria not related to bacteriophages or bacteriophage proteins,
3. detecting bacteria bound to bacteriophages or bacteriophage proteins with a bacterium-specific ligand selected from labeled bacteriophage-specific bacteriophage, labeled bacteriophage-specific bacteriophage protein, bacteriophage-specific bacteriophage coupled to a labeled antibody or a bacteriophage protein coupled to a labeled antibody, and in which said step c) is carried out in the presence of a solution comprising a cation and a protein, or chosen from among a labeled antibody or a labeled aptamer directed against the bacterium.