FR2980213A1 - PROCESS FOR CHARACTERIZING BACTERIA BY DETECTION OF NON-STRUCTURAL PROTEINS OF BACTERIOPHAGES - Google Patents

Abstract

The present invention relates to a method for characterizing at least one bacterium present in a sample, said method comprising at least the following steps: a) obtaining a solution containing at least one bacteriophage capable of being specific for said bacterium (s) ( s) to characterize; b) contacting the solution containing said bacteriophage (s) with said bacterium (s) to be characterized and, optionally, at least one antimicrobial agent; c) To put in conditions allowing bacteriophage infection and multiplication of bacteria within said bacteria; d) Detect by any suitable method the presence of non-structural proteins of bacteriophages, produced during the infection of bacteria; the presence of said non-structural proteins for characterizing said bacterium (s).

Description

A method of characterizing bacteria by detection of non-structural proteins of bacteriophage The technical field of the present invention is that of microbiology. More specifically, the present invention relates to a method for characterizing bacteria, by detecting non-structural proteins of bacteriophages. There are nowadays many methods for detecting and identifying microorganisms and in particular bacteria.

Among these methods, conventional methods of bacterial detection and identification are based on the use of culture media. They require a culture of several hours (12 to 24 hours), in order to obtain a quantity of bacteria necessary to be able to implement the complementary analyzes, generally based on the metabolism of the bacteria.

Molecular biology methods, based on the amplification of bacterial nucleic acids, are now widely used. They are faster and can provide results in less than 5 hours without prior culture; on the other hand, they induce many false-positives and then require a culture step to confirm the results.

Alternative methods based on the use of bacteriophages (or phages) have also been developed. Bacteriophages are bacterial viruses that have multiple bacterial detection interests, such as host cell recognition specificity, rapid self-replication, long shelf life, low cost, and potential to differentiate viable cells from bacterial cells. not viable (linked to the fact that they are only able to infect metabolomically active bacteria). Tailed bacteriophages (Caudoviridae, such as T4 bacteriophage), which are the most commonly encountered, show a cycle that results in the lysis of the infected host bacterium. However, certain bacteriophages, such as filamentous bacteriophages (Inoviridae, such as bacteriophage M13) are released by the infected host bacterium without causing lysis. At present, bacterial detection can be performed by different approaches using bacteriophages. They are described in a recent publication (Smartt and Ripp 2011 [1]) and presented succinctly below. The detection of the bacteria can be carried out: with the aid of a phage component capable of recognizing and binding to a defined target bacterium, such as an endolysin binding domain or a caudal fiber protein. Detection tools are marketed by the applicant under the trademark VIDAS® UP; via phage DNA labeled with a fluorescent dye; using a reporter bacteriophage comprising a reporter gene coding for a bioluminescent (luciferase) or fluorescent (GFP) protein, an enzyme (beta-galactosidase) or an ice nucleation protein. (inaW gene) - which is only expressed in host bacteria infected by the reporter bacteriophage; with the aid of a reporter bacteriophage labeled with a colloidal Quantum-Dot (QD), which is a fluorescent probe consisting of inorganic semiconductor nanocrystals; labeling of bacteriophages requires genetically modifying them to express a capsid protein fused to a peptide that is biotinylated in vivo to interact with a streptavidin functionalized QD (Edgar et al., 2006 [2]; et al 2009 [3]); via signals related to the lysis of the host bacterium consecutive to the phage infection, such as intracellular constituents of the host (enzyme adenylate kinase) or ions released at the time of lysis; using phage amplification, as described in more detail below. When bacteriophages are added to a bacteriophage-susceptible bacteriophage host culture, the infection leads to the formation of many neoformed phage particles that are released into the medium. Different methods are used to detect neoformed phage particles. Among them, the visualization of lysis ranges on culture media in Petri dishes (phage amplification assay) goes through a 4-step protocol: step 1) addition of bacteriophages to the sample and infection of host bacteria if they are present, step 2) before lysis of the host bacteria following the infection, addition of a virucide to inactivate the bacteriophages remained free in the extracellular medium, step 3) neutralization of the virucide and addition of a population of healthy auxiliary helper bacteria (helper or sensor or signal-amplifying cells) which will be rapidly infected by the newly formed bacteriophages resulting from the lysis of the bacteria in the sample, step 4) amplification of the process by infection of the auxiliary bacteria and visualization via the formation of lysis plaques on a bacterial carpet . This method is used by Biotec Laboratories (Ipswich, UK) which markets tests for the detection (FastPlaqueTBTM diagnostic assay) or susceptibility to rifampicin (FastPlaque- ResponseTM diagnostic assay) of Mycobacterium tuberculosis [4,5,6]. An alternative uses an immuno-magnetic separation step (magnetic beads sensitized with an anti-bacteria antibody) of the target cells to avoid the use of virucides (step 2), which can be problematic because of their non-universal efficacy. Immuno-magnetic separation allows the target cells to be concentrated before infection by the bacteriophage and to carry out washes to eliminate bacteriophages remaining free in solution after infection (Favrin et al., 2001 [7]). As an alternative to visualizing the lysis ranges (step 4), the same authors use an optical density (OD) reading of the liquid culture of the auxiliary bacteria (decrease of OD in case of lysis by bacteriophages). This new type of test was used for food matrices artificially contaminated with bacteria (Salmonella enteritidis or Escherichia coli 0157: H7) (Favrin et al., 2003 [8]).

Other adaptations of the phage amplification based assays have been described by detecting either whole phage particles, but bacteriophage constituents such as genomic DNA or structural proteins. Sergueev et al. (2010) [9] described the monitoring of the increase of phage particles neoformed by quantitative PCR in real time using primers targeting the genome of the bacteriophage.

Madonna et al. (2003) [10] described the identification of bacteriophage MS2 capsid protein by MALDI-TOF mass spectrometry for the detection of E. al. In addition, Rees and Voorhees (2005) [11] described the simultaneous MALDI-TOF detection of E. coli. coli and Salmonella following the specific protein signatures (capsid proteins) of their respective bacteriophages. MicroPhage Inc. [12,13,14] has also developed a series of tests for the identification and susceptibility to antibiotics of Staphylococcus aureus based on rapid immunoassay (strip test) detection of newly formed bacteriophage proteins. The latter analysis techniques nevertheless have disadvantages. Indeed, in order not to induce false positives for detection methods coupling phage amplification and mass spectrometry, it is necessary to be able to distinguish the bacteriophages injected to initiate the bacterial infection (parent bacteriophages) of those are produced (newly formed bacteriophages) by the infection of the target bacterium. Thus, WO-A-03/087772 describes the use of tagged bacteriophages (biotinylated capsid protein) for the detection of bacteria. These tagged bacteriophages induce a difference in mass between the parent bacteriophages / markers and the newly formed bacteriophages / markers. This marking makes it possible to use a large quantity of parent bacteriophages, which favors infection in the case where there are few host bacteria present in the biological sample, and thus makes it possible to reduce the limit of detection. In addition, Pierce et al. (2011) [15] describes the use of a 15N stable isotope labeled bacteriophage solution to initiate bacterial infection (parental phage); the labeling of these phages was previously obtained by growing them in a medium containing only the 15N stable isotope. The infection of the target bacterium is in a conventional culture medium composed of 14N and each infected bacterium produces virus particles consisting of protein comprising 14N. Therefore, it is possible to distinguish the parent bacteriophages, whose proteins are labeled with 15N, newly formed bacteriophages, whose proteins contain 14N. Although these techniques effectively prevent false positive results due to the differentiation between the population of bacteriophages used for infection and that of newly formed bacteriophages, they have the disadvantages of having to go through this step of preliminary labeling of the population of bacteriophage used for infection, which is often cumbersome to implement.

Another way of avoiding false-positive results is the use of a quantity of parent bacteriophages (intended for infection of the target bacteria) below the detection limit of the spectrometry instrument. mass. Thus, during the analysis, the bacteriophages detected by mass spectrometry are necessarily bacteriophages neoformed during the bacterial infection. Such a technique is described in Madonna et al. (2003) [10] and Rees and Voorhees (2005) [11]. Madonna et al. (2003) [10] describes a detection method based on the amplification of bacteriophages coupled with MALDI-TOF mass spectrometry. Their methodology comprises three steps: 1) Concentration of bacteria by immunomagnetic separation, 2) Infection of bacteria isolated by a bacteriophage lytic, 3) Detection of amplification of the signal due to the amplification of bacteriophages by MALDI-mass spectrometry TOF.

The bacteriophage used in this study is the MS2 virus, whose genome encodes only 4 proteins. Its lytic viral cycle generates between 10,000 and 20,000 viral particles neoformed in 40 min. The MALDI-TOF spectrum of bacteriophage MS2 (stock solution) 1.109 PFU (Unit Format Range) shows a signal corresponding to the bacteriophage MS2 capsid protein. A 1:10 dilution series made it possible to determine the limit of detection of this marker at 1,106 PFU. Therefore, in their infection protocol the authors use an initial amount of parent bacteriophage of 1.105 PFU less than the detection limit of the marker by the MALDI-TOF mass spectrometer. Thus, the marker signal becomes detectable only if the amplification of the bacteriophage took place following infection of the host bacterium.

Rees and Voorhees (2005) [11] describe a detection method also based on bacteriophage amplification coupled with MALDI-TOF mass spectrometry. Unlike Madonna et al. (2003) [10], this method does not include immunomagnetic separation of bacteria upstream of bacteriophage infection and is not particularly sensitive. On the other hand, it makes it possible to simultaneously detect the presence of two bacterial species present in the same sample by amplifying two bacteriophages, each specific for one of the two species. The signals corresponding to the major capsid proteins of the two bacteriophages are detected by MALDI-TOF. The presence of several species of bacteria and bacteriophages does not induce any particular effects on the detection of markers. The models studied by this team are Escherichia coli / bacteriophage MS2 and Salmonella spp./bacteriophage MPSS-1. 1.105 CFU (Colony Forming Units) / mL of each bacterial species are detected by the method described. The use of a quantity of parent bacteriophages less than the limit of detection of the instrument has the major disadvantage of significantly lengthening the incubation time. Indeed, since the amount of parent bacteriophages is relatively small, it is necessary to prolong the incubation in order to obtain a level of infection of the bacteria such that the quantity of neoformed bacteriophages is greater than the limit of detection of the bacteriophage. instrument. In this context, the objective of the present invention is to provide a method of detecting / characterizing bacteria based on phagic amplification, which is easy to implement and makes it possible to obtain analysis results in times compatible with the needs of analytical laboratories. Thus, the present invention relates to a method for characterizing at least one bacterium, present in a sample, said method comprising at least the following steps: a) obtaining a solution containing at least one bacteriophage capable of being specific to said one or more bacterium (s) to be characterized; b) contacting the solution containing said bacteriophage (s) with said bacterium (s) to be characterized and, optionally, at least one antimicrobial agent; c) To put in conditions allowing bacteriophage infection and multiplication of bacteria within said bacteria; d) Detect by any suitable method the presence of non-structural proteins of bacteriophages, produced during the infection of bacteria; the presence of said non-structural proteins for characterizing said bacterium (s).

By characterization is meant the identification of said bacterium (s) and / or the determination of the potential resistance properties of said bacterium (s) to at least one antimicrobial. The present invention also relates to a method for identifying at least one bacterium present in a sample, said method comprising at least the following steps: a) obtaining a solution containing at least one bacteriophage capable of being specific for said bacterium or bacteria (s) to identify; b) contacting the solution containing said bacteriophage (s) with said bacterium (s) to be identified; c) To put in conditions allowing bacteriophage infection and multiplication of bacteria within said bacteria; d) Detect by any appropriate method the presence of non-structural proteins of bacteriophages, produced during the infection of bacteria; the presence of said nonstructural proteins for confirming the identification of said bacterium (s). The present invention further relates to a method for determining the antimicrobial potential resistance properties of at least one bacterium present in a sample, said method comprising at least the following steps: a) obtaining a solution containing at least one bacteriophage susceptible to be specific for said bacterium (s) whose properties of potential resistance to an antimicrobial must be determined; b) contacting the solution containing said bacteriophage (s) with said bacterium (s) and at least one antimicrobial; c) To put in conditions allowing bacteriophage infection and multiplication of bacteria within said bacteria; d) Detect by any suitable method the presence of non-structural proteins of bacteriophages, produced during the infection of bacteria; the presence of said nonstructural proteins making it possible to confirm the determination of the properties of potential resistance of said bacterium (s) to said at least one antimicrobial. According to a particular embodiment of the invention, the process comprises an additional step c ') of lysis of the bacteria. Indeed, bacteriophages are classified in two populations: - Lytic bacteriophages that have the capacity to lyse the cells they have infected to allow the release of neoformed viral particles, but also non-structural proteins, in the external environment . - Temperate bacteriophages that do not cause lysis of host cells during the release of neo-formed virus particles. These particles are extruded from the bacterial membrane, and the non-structural proteins of the bacteriophage are conserved within the bacterial cell. In the case of this second population, it is therefore necessary to provide in the method according to the invention, a step of lysis of the host bacteria after a time necessary for the infection and the production of the new viral particles, in order to release the non-structural proteins in the external environment and allow their subsequent detection.

Preferably, the detection of the presence of non-structural proteins of bacteriophages is carried out by mass spectrometry. The mass spectrometry used is MS, MS / MS or MS spectrometry followed by MS / MS type spectrometry. Advantageously, the mass spectrometry is an MRM type spectrometry.

According to an alternative embodiment, the detection of the presence of non-structural proteins of bacteriophages can be implemented by a ligand / antiligand reaction. By ligand / anti-ligand reaction is meant a reaction between two specific binding partners. According to a preferred embodiment of the invention, the ligand / anti-ligand reaction is an antigen / antibody reaction. The antigen is constituted by a non-structural protein or one or more peptides derived from said protein, against which antibodies have been produced. Advantageously, the antibody is conjugated to a marker making it possible to demonstrate the antigen-antibody reaction. These so-called immunoassay detection techniques are well known to the biologist and for many years. The detection of nonstructural proteins may for example be carried out by an ELISA (Enzyme-Linked ImmunoSorbent Assay) test.

According to another particular embodiment, the method according to the invention comprises an additional step c ") of concentration or fractionation of the nonstructural proteins, before the detection step.This concentration step makes it possible to increase the sensitivity of detection, insofar as the proteins are isolated from the complex medium in which the lysis has taken place.Adjantageously, the concentration is carried out by means of magnetic beads added to the reaction medium, Such beads may be, by way of example, those marketed by ADEMTECH under the trade name Carboxylic Adembeads.

The fractionation can be carried out, for example, by inserting into the reaction medium antibodies which specifically recognize the bacteriophages present in the medium. These antibodies can be attached to magnetic particles, so that they can be removed from the reaction medium. It is then possible to recover directly in the reaction medium (supernatant) the soluble proteins to which the nonstructural proteins belong. According to another particular embodiment of the invention, the method of the invention comprises an additional step b ') of specific capture of the said bacteria or bacteria prior to the infection step. This specific capture step aims to concentrate the target bacteria and possibly wash them, thus improving the detection limit. Such a specific capture step can thus be performed by immunomagnetic separation (IMS). Such a separation technique is described in particular in document WO-A-03/087772. Another means of specifically capturing the said bacteria or bacteria consists in the use of a phage component as described above (see page 2).

According to another particular mode of the process according to the invention, the non-structural proteins of bacteriophages are taken from the group comprising the proteins d11, motB of bacteriophage T4. Nevertheless, any protein encoded by the genome of the bacteriophage but not included in the composition of the viral particle can also be used. According to another particular mode of the method according to the invention, the identification of said bacterium (s) and / or the determination of the potential resistance properties of said bacterium (s) to at least one antimicrobial, implements at least one peptide of sequence SEQ ID NO: 2, 3, 5 or 6. Advantageously, the bacterium (s) characterized by the process according to the invention are obtained from a culture medium, preferentially liquid. Another subject of the present invention relates to a peptide of sequence SEQ ID No. 2, 3, 5 or 6.

By determining the resistance to at least one antimicrobial means the determination of the resistance of a microorganism to be destroyed by an antimicrobial. Thus, if the microorganism is a bacterium, the antimicrobial against which it can develop resistance is an antibiotic. In the method according to the invention, the detection of nonstructural bacteriophage proteins constitutes an indirect determination of the resistance of the bacteriophage target bacterium to a particular antibiotic. Indeed, when the bacterium has the mechanisms of resistance to said antibiotic, the presence of said antibiotic has no impact on bacterial growth and does not cause the destruction of bacteria. Bacteriophages are therefore able to infect them. It follows therefore a production of non-structural proteins during the multiplication of bacteriophages; said non-structural proteins can then be detected by the method according to the invention. On the other hand, when the bacteria are sensitive to the antibiotic, they are destroyed by the antibiotic, preventing bacteriophage infection of the bacteria. The sample on which the process of the invention may be implemented is any sample likely to contain a target microorganism. The sample may be of biological origin, whether animal, plant or human. It can then correspond to a biological fluid sample (whole blood, serum, plasma, urine, cerebrospinal fluid, organic secretion, for example), a tissue sample or isolated cells. This sample can be used as it is, or can undergo prior analysis, enrichment type preparation, extraction, concentration, purification, culture, according to methods known to those skilled in the art.

The sample may be of industrial origin, or, according to a non-exhaustive list, an air sample, a water sample, a sample taken from a surface, a part or a manufactured product, a food product. Examples of food-based samples include, but are not limited to, a sample of milk products (yogurts, cheeses), meat, fish, egg, fruit, vegetables, water, and beverages (milk , fruit juice, soda, etc.). These food-based samples can also come from sauces or elaborate dishes. A food sample may finally be derived from a feed intended for animals, such as in particular animal meal. Upstream of the detection by mass spectrometry, the sample to be analyzed is preferentially treated beforehand to generate peptides from all the proteins present in the sample in order to fragment these proteins into peptides, for example by digestion with a protein. proteolytic enzyme (protease), or by the action of a chemical reagent. Indeed, the cleavage of the proteins can be done by a physico-chemical treatment, a biological treatment or a combination of the two treatments. Among the treatments that can be used, mention may be made of treatment with hydroxyl radicals, in particular with H 2 O 2. Treatment with hydroxyl radicals causes cleavage of the peptide bonds which occurs randomly on any peptide bond of the protein. The concentration of hydroxyl radicals determines the number of cleavages operated and therefore the length of the peptide fragments obtained. Other chemical treatments may also be used such as, for example, cyanogen bromide (CNBr) treatment which specifically cleaves the peptide bonds at the carboxylic group of the methionyl residues. It is also possible to carry out partial acid cleavage at the aspartyl residues by heating at 1000 ° C. a solution of proteins in trifluoroacetic acid. Protein treatment by enzymatic digestion is nevertheless preferred over physicochemical treatment because it preserves the structure of proteins more, and is easier to control. By "enzymatic digestion" is meant the single or combined action of one or more enzymes under appropriate reaction conditions. Proteolysis enzymes, called proteases, cut proteins at specific locations. Each protease generally recognizes a sequence of amino acids in which it always performs the same cut. Some proteases recognize a single amino acid or a sequence of two amino acids between which they cleave; other proteases only recognize longer sequences. These proteases may be endoproteases or exoproteases. Among the known proteases, as described in document WO-A-2005/098071, mention may be made of: specific enzymes such as trypsin which cleaves the peptide bond at the carboxyl group of the Arg and Lys residues, endolysin which cleaves the peptide bond of the -CO group of lysines, chymotrypsin which hydrolyzes the peptide bond at the carboxylic group of aromatic residues (Phe, Tyr and Trp), pepsin which cuts at the level of the NH2 group of aromatic residues (Phe, Tyr and Trp ), the V8 protease of Staphylococcus aureus strain V8 which cleaves the peptide bond at the carboxylic group of the Glu residue; non-specific enzymes such as thermolysin from the bacteria Bacillus thermoproteolyticus which hydrolyzes the peptide bond of the NH 2 group of hydrophobic amino acids (Xaa-Leu, Xaa-Ile, Xaa-Phe), subtilisin and pronase which are proteases which hydrolyze substantially all the bonds and can transform the proteins into oligopeptides under controlled reaction conditions (enzyme concentration and reaction time). Several proteases can be used simultaneously, if their modes of action are compatible, or they can be used successively. In the context of the invention, the digestion of the sample is preferably carried out by the action of a protease enzyme, for example trypsin. The generation of peptides using a chemical reagent or a protease can be obtained by simple reaction in solution. It can also be implemented with a microwave oven [16], or under pressure [17], or even with an ultrasonic device [18]. In these last three cases, the protocol will be much faster.

Among the peptides thus obtained, the peptides specific for the protein are called proteotypic peptides. These are the ones that will be measured by mass spectrometry.

Similarly, the sample containing proteinaceous characterization markers may also be pretreated for purification purposes. When the markers are of protein origin, this pretreatment purification may be carried out before or after the peptide generation step as described above.

The prior purification of sample treatment is widely known to those skilled in the art and may in particular implement centrifugation, filtration, electrophoresis or chromatography techniques. These separative techniques can be used alone or combined with one another to achieve multidimensional separation. For example, multidimensional chromatography can be used by combining ion exchange chromatography separation with reverse phase chromatography, as described by T. Fortin et al. [19], or H. Keshishian et al. [20]. In these publications, the chromatographic medium may be in column or cartridge (solid phase extraction). The electrophoretic or chromatographic fraction (or the retention time in mono or multidimensional chromatography) of the proteotypic peptides is characteristic of each peptide and the implementation of these techniques therefore makes it possible to select the proteotypic peptide (s) to be assayed. Such a fractionation of the generated peptides makes it possible to increase the specificity and the sensitivity of the subsequent assay by mass spectrometry.

The mass spectrometry to be used in the process of the invention is widely known to those skilled in the art as a powerful tool for the analysis and detection of different types of molecules. In general, any type of molecule that can be ionized can be detected as a function of its molecular mass using a mass spectrometer. Depending on the nature of the molecule to be detected, of protein or metabolic origin, some mass spectrometry technologies may be more suitable. Nevertheless, whatever the mass spectrometry method used for the detection, the latter comprises a step of ionization of the target molecule into so-called molecular ions, in this case a step of ionization of the characterization markers, and a step separation of the molecular ions obtained according to their mass.

All mass spectrometers therefore comprise: i) an ionization source for ionizing the markers present in the sample to be analyzed, that is to say, to give a positive or negative charge to these markers; ii) a mass analyzer for separating ionized markers, or molecular ions, as a function of their mass-to-charge ratio (m / z); iii) a detector for measuring the signal produced either directly by the molecular ions, or by ions produced from the molecular ions, as detailed below. The ionization step necessary for the implementation of mass spectrometry can be carried out by any method known to those skilled in the art. The ionization source makes it possible to bring the molecules to be assayed under a gaseous and ionized state. An ionization source can be used either in positive mode to study positive ions, or in negative mode to study negative ions. Several types of sources exist and will be used depending on the desired result and the molecules analyzed. These include, but are not limited to: - electron ionization (EI), chemical ionization (CI) and chemical desorption ionization (DCI) - fast atom bombardment (FAB), metastable atoms (MAB) or ions (SIMS) , LSIMS) - inductive plasma coupling (ICP) - atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI) - electrospray or electrospray (ESI) - matrix assisted laser desorption ionization ( MALDI), activated by a surface (SELDI) or on silicon (DIOS) - ionization-desorption by interaction with metastable species (DART) In particular, the ionization can be implemented as follows: the sample containing the molecules targets is introduced into an ionization source, where the molecules are ionized in the gaseous state and thus transformed into molecular ions that correspond to the initial molecules. An electrospray ionization source (ESI for ElectroSpray Ionization) makes it possible to ionize a molecule while passing it from a liquid state to a gaseous state. The molecular ions obtained then correspond to the molecules present in the liquid state, with positive mode one, two or even three or more additional protons and therefore carry one, two or even three or more charges. For example, when the target molecule is a protein, an ionization of the proteotypic peptides obtained after fractionation of the target protein, thanks to an electrospray-type source operating in a positive mode, leads to gaseous multicharged ions, with one, two or even three or more additional protons and which therefore carry one, two or even three or more charges, and allows a transition from a liquid state to a gaseous state [21]. This type of source is particularly well suited when the target molecules or proteotypic peptides obtained are previously separated by reverse phase liquid chromatography. Nevertheless, the ionization efficiency of the molecules present in the sample may vary according to the concentration and nature of the different species present. This phenomenon results in a matrix effect well known to those skilled in the art.

A MALDI ionization source will ionize molecules from a solid state sample. The mass analyzer in which is implemented the step of separating the ionized markers according to their mass / charge ratio (m / z) is any mass analyzer known to those skilled in the art. We can mention low-resolution analyzers, quadrupole or quadrupole (Q), 3D (IT) or linear (LIT), also called ion trap, and high-resolution analyzers, which measure the exact mass of the analytes. which use in particular a magnetic sector (FT-ICR) a time-of-flight device (TOF) or an Orbitrap. The separation of the molecular ions as a function of their m / z ratio can be carried out only once (single mass spectrometry or MS), or several successive MS separations can be carried out. When two successive MS separations are performed, the analysis is called MS / MS or MS2. When three successive MS separations are made, the analysis is called MS / MS / MS or MS3 and more generally, when n successive MS separations are made, the analysis is called MS '. Among the techniques implementing several successive separations, the modes SRM (Selected Reaction Monitoring) in case of detection or determination of a single target molecule, or MRM (Multiple Reaction Monitoring) in case of detection or determination of several target molecules, are particular uses of MS2 separation. Similarly MRM3 mode is a particular use of separation in MS / MS / MS. This is called targeted mass spectrometry.

In the case of detection in simple MS mode, it is the mass / charge ratio of the molecular ions obtained which is correlated with the target molecule to be detected. In the case of detection in MS / MS mode, essentially two steps are added, relative to an MS assay, which are: i) fragmentation of the molecular ions, then called precursor ions, to give ions called fragment ions of lere generation, and ii) a separation of ions said fraction ions of the first generation as a function of their mass (m / z) 2, the ratio (m / z) i corresponding to the ratio (m / z) of the precursor ions. It is then the mass / charge ratio of the first generation fragment ions thus obtained which is correlated with the target molecule to be detected. The term "first generation fragment ion" means an ion derived from the precursor ion, following a fragmentation step and whose mass-to-charge ratio m / z is different from the precursor ion. The pairs (m / z) i and (m / z) 2 are called transitions and are representative of the characteristic ions to be detected. The choice of characteristic ions that are detected to be correlated to the target molecule is made by those skilled in the art according to the standard methods. Their selection will lead advantageously to the most sensitive dosages, the most specific and the most robust possible, in terms of reproducibility and reliability. In the methods developed for the selection of proteotypic peptides (m / z) i, and first generation fragment (m / z) 2, the choice is essentially based on the intensity of the response. For more details, see V. Fusaro et al. [22]. Commercial software, such as MIDAS software and MRM Pilot Applied Biosytems or MRMaid [23] can be used by those skilled in the art to enable him to predict all the possible transition couples. It will also be possible to use a database called PeptideAtlas, built by F. Desiere et al. [24] to compile all MRM transitions of peptides described by the scientific community. This PeptideAtlas database is available for free access on the internet. For non-protein molecules, it is also possible to use databases, such as for example that accessible through Cliquid software Applied Biosytems (United States of America). The fragmentation of the selected precursor ions is carried out in a fragmentation cell such as the triple quadrupole type [25], or ion trap type [26], or type of flight time (TOF) [27], which also allow the separation of ions. The fragmentation or fragmentation will be conventionally carried out by collision with an inert gas such as argon or nitrogen, within an electric field, by photoexcitation or photodissociation using an intense light source, collision with electrons or radical species, by applying a potential difference, for example in a flight time tube, or by any other mode of activation. The characteristics of the electric field condition the intensity and the nature of the fragmentation Thus, the electric field applied in the presence of an inert gas, for example in a quadrupole, conditions the collision energy supplied to the ions. This collision energy will be optimized, by the skilled person, to increase the sensitivity of the transition to be assayed. For example, it is possible to vary the collision energy between 5 and 180 e-V in q2 in an AB SCIEX QTRAP® 5500 mass spectrometer from Applied Biosystems (Foster City, USA). Similarly, the duration of the collision step and the excitation energy within, for example, an ion trap will be optimized, by the skilled person, to lead to the most sensitive assay. For example, it is possible to vary this duration, called excitation time, between 0.010 and 50 ms and the excitation energy between 0 and 1 (arbitrary unit) in Q3 in an AB SCIEX mass spectrometer. QTRAP® 5500 from Applied Biosystems. Finally, the detection of the selected characteristic ions is done in a conventional manner, notably thanks to a detector and a treatment system. The detector collects ions and produces an electrical signal whose intensity depends on the amount of ions collected. The signal obtained is then amplified so that it can be processed by computer. A computer set of data processing makes it possible to transform the information received by the detector into a mass spectrum. The principle of the SRM mode, or even the MRM mode, is to specifically select a precursor ion, to fragment it, and then to specifically select one of its fragment ions. For such applications, devices of the triple quadrupole type or triple quadrupole hybrids with ion trap are generally used. In the case of a triple quadrupole device (Q1q2Q3) used in MS2 mode, for the purpose of assaying or detecting a target protein, the first quadrupole (Q1) makes it possible to filter the molecular ions corresponding to the proteotypic peptides characteristic of the protein to be assayed and obtained during a previous step of digestion, according to their mass-to-charge ratio (m / z). Only the peptides having the mass / charge ratio of the desired proteotypic peptide, called ratio (m / z) i, are transmitted in the second quadrupole (q2) and act as precursor ions for the subsequent fragmentation. The q2 analyzer makes it possible to fragment the peptides of mass / charge ratio (m / z) i into first generation fragment ions. Fragmentation is generally achieved by collision of the precursor peptides with an inert gas, such as nitrogen or argon in q2. The first generation fragment ions are transmitted in a third quadrupole (Q3) which filters the first generation fragment ions according to a mass-to-specific charge ratio called ratio (m / z) 2. Only the first generation fragment ions having the mass / charge ratio of a characteristic fragment of the desired proteotypic peptide (m / z) 2 are transmitted in the detector to be detected or even quantified. This mode of operation has a double selectivity, in relation to the selection of the precursor ion on the one hand and the selection of the first generation fragment ion on the other hand. Mass spectrometry in SRM or MRM mode is therefore advantageous for quantification When the mass spectrometry used in the method of the invention is a tandem mass spectrometry (M52, MS3, MS4 or MS5), several analyzers of mass can be coupled together. For example, a first analyzer separates the ions, a collision cell makes it possible to fragment the ions, and a second analyzer separates the fragment ions. Some analyzers, such as ion traps or FT-ICR, are several analyzers in one and can fragment the ions and analyze the fragments directly. The identification may be carried out directly from the sample in which the identification is carried out, or the microorganisms contained in the sample may be cultured by methods well known to those skilled in the art with culture and optimum culture conditions adapted according to the species of microorganisms to be sought, as described by Murray PR et al. in Manual of Clinical Microbiology, 2007, 9th Edition, Vol. I, Section III, Chapter 14, and in particular in Vol. I, Section IV, Chapter 21 for bacteria, Vol. II, Section VI, Chapter 81 for viruses, Vol. II, Section VIII, Chapter 117 for yeast, and Vol. II, Section X, Chapter 134 for protozoa. Instead of culturing the microorganisms, they can be concentrated by capture directly into the sample by means of active surfaces. Such a method has been described by W.-J. Chen et al. [16] that captured different bacterial species using magnetic beads with a Fe304 / TiO2 activated surface. Capture by other means is also possible, such as capture by lectins [28], or by antibodies [29], or by Vancomycin [30]. The capture makes it possible to concentrate the microorganisms and thus reduce or even eliminate the culture step. It saves a lot of time. The identification may also be carried out by mass spectrometry, according to the techniques described above, preferably by MS, by MS / MS, or by MS followed by MS / MS type spectrometry, which constitutes a method of embodiment of the invention. In this case also, the sample may be subjected beforehand to a culture step such as an agar seeding. The use of an identification method by MS is advantageous because it can be achieved in a few minutes and requires a mass spectrometer with a single analyzer, ie a less complex instrument than a spectrometer. Tandem mass used in MS / MS.

The use of an MS identification method followed by MS / MS type spectrometry is also advantageous. It makes it possible to ascertain the identity of the ions observed in MS, which increases the specificity of the analysis. The use of an MRM type MS / MS identification method has the advantage of being more sensitive and simpler than the traditional MS and MS / MS approaches. This method requires neither high-performance software to process the information between the acquisition of the MS spectrum and the MS / MS spectrum, nor change the setting of the machine parameters to link MS and then MS / MS spectra. The MS identification method can be carried out with a raw sample electrospray source, as described by S. Vaidyanathan et al. [31] or by R. Everley et al. [32] after chromatographic separation. Different ranges of m / z then make it possible to identify the microorganisms. S. Vaidyanathan et al. used a window between 200 and 2000 Th and R. Everley et al. a window between 620 and 2450 Th. Mass spectra can also be deconvolved to access the mass of proteins regardless of their state of charge. R. Everley et al. thus exploited the masses between about 5,000 and 50,000 Da. Alternatively, the MS identification method may also be carried out using a MALDI-TOF, as described by Claydon et al [33] and T. Krishnamurthy and P. Ross [34]. The analysis combines the acquisition of a mass spectrum and the interpretation of an expert software. It is extremely simple and can be done in minutes. This identification process is currently spreading in medical analysis laboratories [35]. The identification of bacteria by MS then MS / MS via their proteins present in the sample, has been widely applied by many teams. By way of example, it is possible to cite the recent works of Manes N. et al. [36] who studied the Salmonella enterica peptidome, or the work of R. Nandakumar et al. [37] or L. Hernychova et al. [38] who studied the proteome of bacteria after digestion of proteins with trypsin. The classical approach consists in i) acquiring a spectrum MS, ii) successively selecting each precursor ion observed on the spectrum MS with an intense signal, iii) successively fragmenting each precursor ion and acquiring its spectrum MS / MS, iv) interrogating bases Protein data such as SWISSPROT or NCBI, through software such as Mascot (Matrix Science, London, United Kingdom), BioworksBrowser (Thermo Fisher Scientific) or SEQUEST (Thermo Scientific, Waltham, United States of America), for identify the peptide having a high probability of corresponding to the observed MS / MS spectrum. This method can lead to the identification of a microorganism if a protein or peptide characteristic of the species is identified. One of the advantages of the use of mass spectrometry is that it is particularly useful for quantifying molecules, in this case markers of the typing properties, resistance to at least one antimicrobial. To do this, the current intensity detected is used, which is proportional to the amount of target molecule. The current intensity thus measured may serve as a quantitative measure for determining the quantity of target molecule present, which is characterized by its expression in units of the International System (SI) of type mol / m3 or kg / m3, or by the multiples or submultiples of these units, or by the usual derivatives of the SI units, including their multiples or submultiples. By way of nonlimiting example, units such as ng / ml or fmo1 / 1 are units characterizing a quantitative measurement. Calibration is nevertheless necessary in order to be able to correlate the area of the peak measured, corresponding to the intensity of current induced by the ions detected, with the quantity of target molecule to be assayed. For this purpose, the calibrations conventionally used in mass spectrometry can be implemented in the context of the invention. MRM assays are typically calibrated using external standards or, preferably, using internal standards as described by T. Fortin et al. [19]. In the case where the target molecule is a proteotypic peptide, which makes it possible to assay a protein of interest, the correlation between the quantitative measurement and the quantity of target proteotypic peptide, and subsequently of the protein of interest, is obtained by calibrating the signal measured with respect to a standard signal for which the quantity to be determined is known. Calibration can be performed by means of a calibration curve, for example obtained by successive injections of standard prototypic peptide at different concentrations (external calibration), or preferentially by internal calibration using a heavy peptide, as standard internal, for example according to the AQUA, QconCAT or PSAQ methods detailed below. By "heavy peptide" is meant a peptide corresponding to the proteotypic peptide, but in which one or more carbon atoms 12 (12C) is (are) replaced by carbon 13 (13C), and / or one or more Nitrogen atoms 14 (14N) are (are) replaced by nitrogen (15N). The use of heavy peptides, as internal standards (AQUA), has also been proposed in the patent application US 2004/0229283. The principle is to artificially synthesize proteotypic peptides with amino acids with heavier isotopes than the usual natural isotopes. Such amino acids are obtained, for example, by replacing some of the carbon atoms 12 ('2C) with carbon 13 (13C), or by replacing some of the nitrogen atoms 14 (14N) with nitrogen ( 15N). The artificial peptide (AQUA) thus synthesized has the same physicochemical properties as the natural peptide (with the exception of a higher mass). It is generally added, at a given concentration, to the sample, upstream of the mass spectroscopy assay, for example between the treatment resulting in the cleavage of the proteins of the sample of interest and the fractionation of the peptides obtained after the analysis. treatment stage. As a result, the AQUA peptide is co-purified with the natural peptide to be assayed, during the fractionation of the peptides. The two peptides are therefore injected simultaneously into the mass spectrometer for the assay. They then undergo the same ionization yields in the source. The comparison of the peak areas of the natural peptides and AQUA, the concentration of which is known, makes it possible to calculate the concentration of the natural peptide and thus to go back to the concentration of the protein to be assayed. A variant of the AQUA technique has been proposed by J.-M. Pratt et al. [39] under the name of QconCat. This variant is also described in the patent application WO 2006/128492. It consists of concatenating different AQUA peptides and producing the artificial polypeptide as a heavy recombinant protein. The recombinant protein is synthesized with amino acids having heavy isotopes. In this way, it is possible to obtain a standard for calibrating the simultaneous determination of several proteins at a lower cost. The QconCAT standard is added from the beginning, upstream of the treatment leading to the cleavage of the proteins and before the steps of protein fractionation, denaturation, reduction and then blocking of the thiol functions of the proteins, if these are present. The QconCAT standard thus undergoes the same treatment cycle leading to cleavage of proteins as the natural protein, which allows to take into account the yield of the treatment step leading to cleavage of proteins. Indeed, the treatment, in particular by digestion, of the natural protein may not be complete. In this case the use of an AQUA standard would underestimate the amount of natural protein. For an absolute dosage, it may therefore be important to consider processing efficiencies leading to protein cleavage. However, V. Brun et al. [40] showed that sometimes the QconQAT standards did not exactly reproduce the processing efficiency, particularly by digestion of the natural protein, probably because of a different three-dimensional conformation of the QconCAT protein. V. Brun et al. [40] then proposed using a method called PSAQ and described in the patent application WO 2008/145763. In this case, the internal standard is a recombinant protein, having the same sequence as the natural protein but synthesized with heavy amino acids. The synthesis is carried out ex-vivo with heavy amino acids. This standard has the exact same physicochemical properties as the natural protein (except for a higher mass). It is added from the beginning, before the protein fractionation step, when the latter is present. It is therefore co-purified with the native protein during the protein fractionation step. It has the same processing yield, especially by digestion, as the native protein. The heavy peptide obtained after cleavage is also co-purified with the natural peptide, if a peptide fractionation step is performed. The two peptides are therefore injected simultaneously into the mass spectrometer, for quantitative determination. They then undergo the same ionization yields in the source. The comparison of the peak areas of the natural peptides and the reference peptides in the PSAQ method makes it possible to calculate the concentration of the protein to be assayed taking into account all the steps of the assay procedure. All of these techniques, namely AQUA, QconCAT or PSAQ or any other calibration technique, used in mass spectrometry assays and in particular in the MRM or MS assays, can be implemented to carry out the calibration, in According to the invention, the method for characterizing bacteria employs at least one specific bacteriophage of the bacterium to be characterized (identification and / or properties of potential resistance to one or more antimicrobials). For example, the characterization of Escherichia coli B can be carried out using bacteriophage T4. In particular, the characterization of Escherichia coli B can be carried out by detecting T4 phage MI and motB proteins, which are nonstructural proteins. For the dII protein, the characterization of Escherichia coli B can carry out the complete protein of sequence SEQ ID No. 1 below: MIKQLQHALELQRAWNNGHENYGASIDVEAEALEILRYFKHLNPAQTAL AAELQEKDELKYAKPLASAARKAVRHFVVTLK It can also use at least one peptide belonging to the protein dII of SEQ ID No. 2 and 3, as defined in Table 2, hereinafter: Peptide SEQ ID Amino Acid Sequence Localization in SEQ ID NO: 1 2 QLQHALELQR 4-13 3 HLNPAQTALAAELQEK 42-57 Table 2 20 For the protein motb, characterization of Escherichia coli B may implement the full length protein of SEQ ID NO 4 following sequence: MIINIGELARVSDKSRSKAAGKLVEVVSIQLKHGVKDEDSEVKVRIIPKDG KSKPQFGYVRAKFLESAFLKAVPAKGIETIDTSHVGVDFKWKLGQAIKFIA 25 PCEFNFIKDDGRVVYTRAMCGYITDQWVEDGVKLYNVVFLGTYKVIPES WIKHYSNALYA It can implement at least one peptide belonging to the motb protein of SEQ ID NO: 5 and 6, as defined in Table 3, hereinafter: Pepti SEQ ID NO: 4 MIINIGELAR 1-10 6 LYNVVFLGTYK 136-146 5 Table 3 Of course, at least one peptide means at least one, at least two, at least one peptide. three, at least four, at least five, at least 6 or more representative peptides of the marker to be detected. Preferably, at least two or even at least three or even at least four peptides per characteristic will be used. Peptides useful for the purposes of the invention, of sequence SEQ ID No. 2, 3, 5, 6, are new and constitute another subject of the invention. The method of the invention and its advantages will emerge from the rest of this description relating various nonlimiting examples of implementation of the method of the invention. Example 1: Identification of non-structural proteins of bacteriophage T4 as markers of bacterial infection Protein markers of infection were identified by centrifuging infected bacteria not yet lysed. Indeed, during the stages of the viral cycle that precede lysis, the phage proteins are all concentrated within the host bacterium. The bacterial pellet is then chemically lysed (ethanol / formic acid protocol, adapted from Freiwald and Sauer, S. Phylogenetic classification and identification of bacteria by mass spectrometry, Nat Protoc 4, 2009). After analysis by liquid chromatography coupled to high-resolution mass spectrometry (LC-MS) (Orbitrap type), protein markers of the infection of E. coli. coli B by bacteriophage T4 could be identified: structural and nonstructural proteins. Liquid Chromatography Parameters: - Accela Chromatographic Chain (Thermo Fisher Scientific) - Zorbax C18-300SB column, 2.lmm inner diameter, 150mm long, 5itm particle size (Agilent Technologies) - Solvent A: H2O - 0.1% acid formic acid - Solvent B: ACN - 0.1% formic acid - Gradient HPLC defined in Table 4 below: Time (min) Flow rate (μL / min) Solvent A (%) Solvent B (%) 0 200 95 5 40 200 40 60 45 200 5 95 48 200 5 95 50 200 95 5 60 200 95 5 Table 4 The eluate at the end of the chromatographic column is directly injected into the electrospray ionization source (ESI) of the LTQ Orbitrap Discovery mass spectrometer (Thermo). Fisher Scientific). The machine parameters used are as follows: Polarity: positive Electrospray source (ESI) Cone voltage: 4000V Nebulization gas: 35 (arbitrary unit) Gas curtain: 10 (arbitrary unit) Auxiliary gas: 20 (arbitrary unit) - Sweep type : Sweep depending on acquisition data - Scanning event 1 Dynamic range (m / z): 200-2000 Resolution: 30000 Polarity: positive - Scanning event 2: Smart scanning with MS / MS of the most intense ions (repeated for the three most intense)) Type of collision: CID Minimum required signal: 500 Energy of collision: 35 State of charge by default: 2 This analysis made it possible to identify a large number of bacteriophage proteins.

Of these, many are nonstructural proteins. The first identified as potentially interesting for use in the characterization of E. coli B is the motB protein, a transcriptional regulatory protein. It is one of the first non-structural proteins identified by the Bioworks Browser software (V3.3.1 SP1, Thermo Fisher Scientific). The second is the low molecular weight (<10kDa) protein dll, which allows it to be detected as intact (undigested) protein or as proteotypic peptides. The following examples relate only to these two nonstructural proteins. It could nevertheless be envisaged to use other proteins identified by the Bioworks software for the E. coli B / bacteriophage T4.25 model. Example 2: E. coli B bacteria protocol by detection of non-structural proteins of bacteriophage T4 1- From a culture of E. neck / B in exponential growth phase in a liquid LB medium, add the bacteriophages T4 with a multiplicity of infection equal to 0.01 in the presence of a protease inhibitor (pefabloc to 0.25 mM final). 2- Incubate 2h at 37 ° C with stirring at 22Orpm. 3- Centrifuge 20min at 12000g at 4 ° C. 4- Recover the lysis supernatant. 5- Take 40 μL of magnetic beads of carboxyl-Adembeads type (Ademtech), magnetize the beads and discard the supernatant. 6- Wash the beads with 200 μL of 100mM sodium acetate (pH = 5.5), magnetize the beads and discard the supernatant. Repeat this step a second time. Resuspend the beads in 400 ml of 100mM sodium acetate (pH = 5.5). 8- Add 2001.11, lysis supernatant prepared in step 4. Mix well, and let stand a few minutes on the bench. 9- Feel the balls and discard the supernatant. 10- Wash the beads with 2001.1.L of 100mM sodium acetate (pH = 5.5), magnetize the beads and discard the supernatant. Repeat this step a second time. 11- Elute nonstructural proteins with 401.11 of 2% formic acid. Thoroughly homogenize, and let rest a few minutes on the bench. 12- Feel the beads and recover the supernatant (or eluate). 13- Take 20 μl of the eluate prepared in step 12, and neutralize with 30 μl of 0.4 M ammonium bicarbonate buffer (check that p1-174). 14- Add 1p, g of trypsin (Promega) 15-Digestion at 37 ° C overnight at 500rpm (Thermomixer Eppendorf). 16- Fractionate the digestate prepared in step 15 on a 10kDa filtration unit by centrifugation for 10 min at 10000 g (Nanosep, membrane omega 101c1Da). Recover the filtrate. Further information on the protocol: Infection: Infection of host bacteria by bacteriophages is carried out in liquid medium (LB medium) with a multiplicity of infection equal to 0.01 (i.e. a ratio of the number bacteriophages on the number of bacteria equal to 0.01, ie 1 bacteriophage per 100 bacteria). The incubation is carried out for 2 h at 37 ° C. with stirring at 22Orpm. The infection is carried out in the presence of a protease inhibitor (pefabloc at 0.25mM) in order to keep intact nonstructural proteins in the lysis supernatant. A short centrifugation at 12000 g eliminates cell debris and recover the lysis supernatant.

Concentration: Subsequently, these nonstructural proteins are concentrated on magnetic carboxylic beads (Carboxyl-Adembeads, Ademtech). A first step of conditioning the beads is carried out in order to deprotonate the carboxylic groups, and thus obtain C00- groups to carry out cation exchange, more specifically positively charged proteins. Following this conditioning, the sample is loaded onto the cation exchange beads. The positively charged proteins are captured, then washed, and finally eluted at acidic pH (pH to reprotonate the carboxylic groups).

Digestion: Enzymatic digestion with trypsin (Promega) makes it possible to generate proteotypic peptides. The neutralization of the eluate of the magnetic beads is necessary because the enzymatic activity of trypsin is optimal in a buffer whose pH is between 7 and 9.

Fractionation: The digestate obtained is fractionated on a 10kDa membrane by centrifugation (Nanosep 10kDa, Pall Corporation). The filtrate, containing the proteotypic peptides, is then analyzed by liquid chromatography coupled to mass spectrometry in MRM mode. Example 3: Detection of the Markers in the Lysis Supernatant (T4 phage model) Each sample is treated according to Example 2 , then a volume of 20gt of digested proteins is injected and analyzed according to the following conditions: Agilent 1100 chromatographic chain (Agilent Technologies, Massy, France) Zorbax C18-300SB column, 2.1mm internal diameter, 150mm long, 5itm particle size (Agilent Technologies) Solvent A: H2O - 0.1% formic acid Solvent B: ACN - 0.1% formic acid HPLC gradient defined in Table 5 below: Time (min) Flow (μL / min) Solvent A (%) Solvent B ( %) 0 200 95 5 2 200 95 5 13 200 30 70 14 200 10 90 200 10 90 16 200 95 5 22 200 95 5 Table 5 Synthetic peptides of sequences identical to SEQ ID NO 2, 3, 5 and 6 , labeled with stable isotopes 13C and 15N lysine or terminal arginine (the trypsic digestion inducing cut in R / K terminal) are added to the sample. These internal standards make it possible to standardize the analytical variations of liquid chromatography and the ionization process, but also to ensure the identification of proteotypic peptides. The eluate leaving the chromatographic column is directly injected into the ionization source of the TSQ Quantum Ultra type mass spectrometer (Thermo Fisher Scientific). The peptides, resulting from the digestion of proteins previously isolated on magnetic beads, are then analyzed by the mass spectrometer in MRM mode. Only the peptides indicated in the following table are detected. For this, the fragments (from a CID type collision) indicated in Table 6 below, are detected.

Protein Transition Peptide Position Ions Fragments # 1 dII QLQHALELQR 4-13 y4 monocharged 2 dII QLQHALELQR 4-13 y5 monocharged 3 dII HLNPAQTALAAELQEK 42-57 y6 monocharged 4 dII HLNPAQTALAAELQEK 42-57 y7 monocharged 5 motB MIINIGELAR 1-10 y5 monocharged 6 motB MIINIGELAR 1-10 y8 monocharged 7 motB LYNVVFLGTYK 136-146 y7 monocharged 8 motB LYNVVFLGTYK 136-146 y9 monocharged Table 6 The state of charge of the precursor peptide, its retention time and the transitions, that is to say the ratios (m / z) at Q1 and (m / z) at Q3 are shown in Table 7. The collision energy used to fragment the precursor ion and the value of the "tube lens" to transfer fragment ions are also indicated. Transition n ° Filtered wire time in collision energy Tube State, filtered retention in Q3 lens charge d of precursor Q1 1 3 8.2 412.8 545.1 16 68 2 3 8.2 412.8 658.2 18 78 3 3 9.1 578.8 717.2 17 73 4 3 9.1 578.8 788.3 20 70 5 2 10.2 565.5 545.1 23 78 6 2 10.2 565.5 885.2 23 87 7 2 10.3 659.0 827.3 21 75 8 2 10.3 659.0 1040.5 22 80 15 Table 7 The other machine parameters used are as follows: Scan type: MRM Polarity: positive Ionization source: - Electrospray ionization (ESI) 5 - Cone voltage: 4000 V - Nebulization gas: 60 (arbitrary unit) - Curtain gas: 2 (arbitrary unit) - Auxiliary gas: 15 (arbitrary unit) - Temperature of source: 350 ° C 10 - Source collision energy: 5V Resolution Ql / Q3: 0.7 (fwhm) Total cycle time = ls The areas obtained for each of the transitions were measured. All transitions whose area is greater than 1000 (arbitrary unit) are considered positive and are marked "1" in Table 8 below. All transitions whose area is less than 1000 are considered negative and are marked "0" in Table 8 below. Example 4 Preparation of the bacteriophage T4 stock solution The T4 bacteriophage stock solution used as parents is prepared according to the protocol described below: 1- From a liquid culture of E. coli B in exponential growth phase, add T4 bacteriophages with a multiplicity of infection equal to 0.01 in the presence of a protease inhibitor (pefabloc at 0.25 mM final). 2- Incubate 2h at 37 ° C with stirring at 22Orpm. 3- Centrifuge 20min at 12000g at 4 ° C. 4- Recover the lysis supernatant. 5- Centrifuge the lysis supernatant at 12000g and 4 ° C overnight. 6- Resuspend the pellet with TSG buffer (0.01M Tris-HCl pH7.5 / 150 mM NaCl / 0.03% gelatin). 7- Store the bacteriophage stock solution at 4 ° C.

When the non-structural protein capture protocol described in Example 1 is applied to this bacteriophage stock solution (storage time at 4 ° C. greater than 1 year), no signal is detected for the MI and motB proteins. (transitions 1-8), as shown in Table 8. Therefore, the detection of nonstructural markers (absent from the bacteriophage stock solution) allows the use of parent bacteriophage concentrations that may be greater than the detection limit of the instrument. Transition # Protein Supernatant of lysis stock Solution 1 dII 1 0 2 dII 1 0 3 dII 1 0 4 dII 1 0 5 motB 1 0 6 motB 1 0 7 motB 1 0 8 motB 1 0 Table 8 Bibliography [1] Smartt, AE & Ripp, S.Anal Bioanal Chem 400, 991-1007 (2011). [2] Edgar, R. et al. Proc. Natl. Acad. Sci. U.S.A 103, 4841-4845 (2006). [3] Yim, P.B. et al. Biotechnol. Bioeng 104, 1059-1067 (2009). [4] Biotec FAST plate-ResponseTM assay at <http://www.biotec.com/pdf/FASTPlaque-Response Lab sheet Rev 1.pdf> [5] Wilson, SM, al-Suwaidi, Z., McNerney, R Porter, J. & Drobniewski, F. Nat. Med. 3, 465-468 (1997). [6] Wilson, S.M. & Biotec Diagnostics. Method to detect bacteria (1997). [7] Favrin, S.J., Jassim, S.A. & Griffiths, M.W. Appl. About. Microbiol. 67, 217224 (2001). [8] Favrin, S.J., Jassim, S.A. & Griffiths, M.W. Int. J. Food Microbiol 85, 63-71 (2003). [9] Sergeev, K.V., He, Y., Borschel, R.H., Nikolich, M.P. & Filippov, A.A. PLoS ONE 5, e11337 (2010). [10] Madonna, A.J., Van Cuyk, S. & Voorhees, K.J. Rapid Commun. Mass Spectrom 17, 257-263 (2003). [11] Rees, J.C. & Voorhees, K.J. Rapid Commun. Mass Spectrom 19, 2757-2761 (2005). [12] MicroPhage Inc. (Longmont, CO), at <http://www.microphage.com/technology/> [13] MicroPhage Inc. (Longmont, CO), MRSA / MSSA Blood Culture Test, at <http: //www.microphage.com/product/bloodMrsaMssaUS. cfm? CFID = 3839487 & CFT 0 KEN = 41d40c704f596964-5A2D4401-EF5F-D568-DO5C4C35AF5E113C> [14] Bhowmick, T. et al. Poster ASM 2011. [15] Pierce, C.L., Rees, J.C., Fernandez, F.M. & Barr, J.R. Anal. Chem 83, 22862293 (2011). [16] W.-J. Chen et al., 2008, Anal. Chem., 80: 9612-9621 [17] D. Lopez-Ferrer et al., 2008, Anal. Chem., 80: 8930-8936 [18] D. Lopez-Ferrer et al., 2005, J. Proteome res., 4 (5): 1569-1574 [19] T. Fortin et al., 2009, Mol. . Cell Proteomics, 8 (5): 1006-1015. [20] H. Keshishian et al., 2007, Mol. Cell Proteomics, 2212-2229. [21] Gaskell, Electrospray: Principles and Practice, 1997, J. Mass Spectrom., 32, 677688). [22] V. Fusaro et al., 2009, Nature Biotech. 27, 190-198. [23] J. Mead et al., Nov. 15, 2008, Mol. Cell Proteomics, E-pub. [24] F. Desiere et al., 2006, Nucleic Acids Res., 34 (database issue): D655-8). [25] L. Anderson & C. Hunter, 2006, Mol. Cell Proteomics, 573-588). [26] B. Han & R. Higgs, 2008, Brief Funct Genomic Proteomic., 7 (5): 340-54). [27] K.-Y. Wang et al., 2008, Anal Chem, 80 (16) 6159-6167). [28] J. Bundy & C. Fenselau, 1999, Anal. Chem. 71: 1460-1463. [29] K-C Ho et al., 2004, Anal. Chem. 76: 7162-7268. [30] Y.S. Lin et al., 2005, Anal. Chem., 77: 1753-1760. [31] S. Vaidyanathan et al., 2001, Anal. Chem., 73: 4134-4144. [32] Everley R. et al., 2009, J. Microbiol. Methods, 77: 152-158. [33] Claydon et al., 1996, Nature Biotech. 14: 1584-1586. [34] T. Krishnamurthy & P. Ross, 1996, Rapid Com. Mass Spec., 10: 1992-1996. [35] P. Seng et al., 2009, Clin. Infect. Dis., 49: 543-551. [36] Manes N. et al., 2007, Mol. & Cell. Proteomics, 6 (4): 717-727. [37] R. Nandakumar et al., 2009, Oral Microbiology Immunology, 24: 347-352). [38] L. Hernychova et al., 2008, Anal. Chem., 80: 7097-7104. [39] J.-M. Pratt et al., 2006, Nat. Protoc, 1: 1029-1043. [40] V. Brun et al., 2007, Mol. Cell Proteomics, 2139-2149.

Claims (15)

REVENDICATIONS1. A method of characterizing at least one bacterium, present in a sample, comprising identifying said bacterium (s) or determining the potential resistance properties of said bacterium (s) to at least one antimicrobial, said method comprising at least the following steps: a) obtaining a solution containing at least one bacteriophage capable of being specific to said bacterium (s) to be characterized; b) contacting the solution containing said bacteriophage (s) with said bacterium (s) to be characterized and, optionally, at least one antimicrobial agent; c) To put in conditions allowing bacteriophage infection and multiplication of bacteria within said bacteria; d) Detect by any suitable method the presence of non-structural proteins of bacteriophages, produced during the infection of bacteria; the presence of said non-structural proteins making it possible to confirm the identification of said bacterium (s) or to confirm the potential resistance of said bacterium (s) to at least one antimicrobial. 2. Method according to claim 1, comprising an additional step c ') of lysis of the bacteria. 3. Method according to claim 1 or 2, wherein the detection of the presence of non-structural proteins of bacteriophages is carried out by mass spectrometry. 4. Method according to the preceding claim, wherein the mass spectrometry is an MS, MS / MS or MS type spectrometry followed by MS / MS type spectrometry. 5. Method according to the preceding claim, wherein the mass spectrometry is an MRM type spectrometry. 6. The method of claim 1 or 2, wherein the detection of the presence of non-structural proteins of bacteriophages is carried out by a ligand / anti-ligand reaction. 7. Method according to the preceding claim, wherein the ligand / anti-ligand reaction is an antigen / antibody reaction. 8. Method according to one of the preceding claims, comprising an additional step c ") of concentration of nonstructural proteins, before the detection step. 9. Process according to the preceding claim, in which the concentration step is carried out by means of magnetic beads. 10. Method according to one of the preceding claims, comprising an additional step b ') of specific capture of said bacteria before the infection stage. 11. Method according to the preceding claim, wherein the specific capture is carried out by immunomagnetic separation (IMS). 12. Method according to one of the preceding claims, wherein the non-structural proteins of bacteriophages are taken from the group comprising the proteins dut motB bacteriophage T4. 13. Method according to one of the preceding claims, wherein the identification of said bacterium (s) and / or the determination of the potential resistance properties of said bacterium (s) to at least one antimicrobial, sets at least one peptide of sequence SEQ ID No. 2, 3, 5 or 6. 14. Method according to one of the preceding claims, wherein said bacterium (s) (s) are obtained from a culture medium. 15. Peptide of sequence SEQ ID No. 2, 3, 5 or 6. 10