PROCEDURE FOR DETERMINATION OF METHICILLIN RESISTANCE OF STAPHYLOCOCCUS AUREUS STRAINS

Abstract

The present invention relates to a method for determining the methicillin resistance properties of a strain of Staphylococcus aureus present in a biological sample, comprising the following steps: a) incubation of the biological sample containing said strain of Staphylococcus aureus for at least 15 minutes, in the presence of a beta-lactam antibiotic selected from the following group: cefoxitin and 6-APA (6-aminopenicillanic acid), b) isolation of the bacteria present in said biological sample, c) lysis of the bacteria and hydrolysis of the bacterial proteins, in order to obtain a mixture of peptides, d) analysis of this mixture of peptides by targeted mass spectrometry, the detection of at least one peptide from the PBP2a protein (SEQ ID NO. 1) or PBP2c protein (SEQ ID NO. 2) during this analysis step being indicative of the methicillin resistance of the strain of Staphylococcus aureus present in said biological sample.

Description

Title of the invention: METHOD FOR DETERMINING METICILLIN RESISTANCE OF STAPHY-LOCOCCUS AUREUS STRAIN FIELD OF INVENTION

[0001] The present invention relates to a method for characterizing bacterial strains belonging to the staphylococcus family, and in particular to a method for identifying bacterial strains resistant to certain antibiotics. This allows healthcare professionals to select appropriate antibiotics for each infected patient, based on this resistance data. STATE OF THE ART

[0002] The increased use of antibiotics in recent decades has led to the emergence of resistance mechanisms in many bacterial species.

[0003] The first strains of Staphylococcus aureus (golden staph) resistant to penicillin were characterized only two years after the introduction of this antibiotic into the chain of treatment of bacterial infections (Kirby, 1944) and spread rapidly, leading to the gradual cessation of the use of penicillin in clinical practice in favor of new semi-synthetic antibacterial molecules such as methicillin and oxacillin.

[0004] Then in the 1960s, new strains of Staphylococcus aureus resistant to methicillin, but also to other antibiotic compounds, appeared. These strains are designated by the acronym MRSA (Methicillin Resistant Staphylococcus aureus).

[0005] These MRSA strains express a particular toxin, LPV or Panton-Valentine leukocidin. This so-called "porogen" toxin induces pores in cell walls, particularly those of immune system cells, thus killing cells capable of fighting the infection.

[0006] Although initially confined to hospital settings, MRSA strains have since spread throughout the population. In 2019, in Europe, it was estimated that MRSA strains accounted for between 1% and 46% of Staphylococcus aureus infections. According to the World Health Organization, a person infected with an MRSA strain has a 64% higher probability of death than a person infected with a non-resistant strain.

[0007] The detection of MRSA strains is therefore a major issue for the management and treatment of patients with Staphylococcus aureus infections.

[0008] The MRSA phenotype is due to the expression of a specific protein, PBP2a for "Penicillin binding protein 2a", encoded by the MecA gene, and in rarer cases, the expression of the PBP2c protein encoded by the MecC gene. These proteins They "replace" the transpeptidase PBP2, an enzyme involved in bacterial cell wall formation. Since the alternative proteins PBP2a and PBP2c have a low affinity for methicillin and penicillin, the presence of these antibiotics does not inhibit their activity, and cell wall synthesis continues normally, thus ensuring bacterial growth even in the presence of these antibiotics.

[0009] Since the appearance of the first MRS A strains, several methods for detecting this resistance property have been developed, based on three main approaches: - Phenotypic methods are based on an evaluation of bacterial growth in the presence of antibiotics; - Molecular biology methods are based on the detection of MecA and / or MecC genes in the bacterial genome of the strains studied; Immunological and proteomic methods are based on the detection of the PBP2a protein or its peptides. Among these technologies, immunochromatography (detection using antibodies) and mass spectrometry are particularly used.

[0010] These different approaches are briefly presented below.

[0011] Phenotypic methods

[0012] Traditional agar diffusion methods, although old, are still widely used in laboratories due to their simplicity and low cost. They consist of culturing the bacteria on agar in the presence of an antibiotic disc, usually oxacillin or cefoxitin. The susceptible or resistant phenotype of the bacterial strain is determined by evaluating the diameter of the growth inhibition zone around the disc.

[0013] The main disadvantage of this technology is the time required, approximately 12 to 18 hours, to obtain the result (Felten, 2002).

[0014] The arrival on the market of instruments such as the Vitek® 2 distributed by BioMérieux, or the Phoenix™ distributed by Becton Dickinson, has enabled the high-throughput automation of antibiotic susceptibility testing. Although more robust than agar diffusion methods, these tests are also limited by the bacterial growth time. In the case of Staphylococcus aureus, the result is obtained in approximately 10 to 13 hours.

[0015] In addition, this technology is sensitive to factors that can influence bacterial growth such as inoculum volume, incubation time and temperature, medium, pH or salt concentration (Tenover et al., 1999).

[0016] Molecular biology methods

[0017] The advent of molecular biology methods has significantly improved the time required to identify MRSA strains compared to phenotypic methods. The high sensitivity and specificity of currently marketed tests make detection of the MecA gene by PCR (Polymerase Chain Reaction) the gold standard for MRSA diagnosis. Commonly used tests include geneXpert MRSA / SA BC distributed by Cepheid, GeneOhm™ StaphSR Assay distributed by Becton Dickinson, eazyplex®MRSAplus distributed by AmplexDiagnostics, and FilmArray BCID distributed by bioMérieux. These tests allow for the detection of MRSA strains in 1 to 3 hours, directly from a positive blood culture bottle, eliminating the need for prior subculture steps involving isolating and proliferating the bacteria.

[0018] The main disadvantage of this technology is that it tends to generate "false positives", particularly in the case of strains carrying an SSCmec cassette not containing the MecA gene, or for strains expressing a variant protein encoded by a gene with a slightly different sequence from that established for the MecA gene.

[0019] Moreover, the high cost of molecular biology analysis remains a major obstacle to their systematic use.

[0020] Immunological tests: detection of the PBP2A protein

[0021] The use of latex beads coupled with a specific monoclonal antibody Detecting the PBP2a protein allows for visualization of agglutination with the naked eye, indicating the presence of the antigen in the sample. This is the principle behind the PBP2' Oxoid tests distributed by Thermo Fisher Scientific and the Mastalex-MRSA tests distributed by Mast Diagnostic.

[0022] The duration of the test itself is between 15 and 20 minutes. This test has very good performance with a sensitivity of 100% and a specificity greater than 99%.

[0023] The major disadvantage of this technique is that it requires a minimum quantity of 1.5 x 109 bacteria in the sample tested, which implies a bacterial culture step lasting at least 10 hours.

[0024] Furthermore, these tests do not allow the detection of MRSA strains expressing the PBP2c protein, which is not recognized by some of the monoclonal antibodies used. This has notably been shown for the PBP2' Oxoid test (Dupieux et al., 2017).

[0025] Immuno-chromatographic tests have also been marketed for the detection of MRSA strains, for example the Clearview™ PBP2a SA Culture Colony Test kit distributed by Alere.

[0026] Although very rapid and possessing a sensitivity and specificity greater than 98%, this test also requires a bacterial culture step.

[0027] The Clearview™ PBP2a SA Culture Colony Test can advantageously detect PBP2c is detected from colonies growing at the edge of a cefoxitin disc after culture on agar. This induction step, necessary for the detection of MecC, however, results in an additional delay of approximately 18 hours before the results are available (Dupieux et al., 2017).

[0028] Targeted mass spectrometry

[0029] Recent studies have highlighted the potential of so-called "bottom-up" proteomic approaches, i.e. based on peptide fragmentation spectra by targeted mass spectrometry, for the identification of pathogens and the highlighting of resistance mechanisms.

[0030] These approaches include the following steps: 1) Mechanical or chemical lysis of the bacterial sample to release intracellular proteins, 2) Enzymatic digestion of bacterial proteins using proteases (typically trypsin) to generate a mixture of peptides 3) Chromatographic separation coupled with targeted mass spectrometry analysis of the sample, where the mass spectrometer operates in MS / MS mode for the detection of marker peptides of resistance proteins 4) Reprocessing the results to validate the presence / absence of marker peptides in the sample.

[0031] Targeted mass spectrometry has been used successfully to characterize the resistance and virulence of Staphylococcus aureus strains by detecting in particular PBP2a or virulence factors such as PVL (Charretier et al., 2015).

[0032] International application WO 2011 / 045544 describes a method comprising the characterization of the resistance and virulence of Staphylococcus aureus by liquid chromatography coupled with targeted mass spectrometry, where the starting biological material is a colony isolated on agar medium.

[0033] Finally, it has recently been shown that the PBP2a protein can be detected in its intact form after separation by liquid chromatography and MS analysis (a "top-down" technology based on the analysis of whole-protein fragmentation spectra). By eliminating the enzymatic digestion step of the protein, the time required to obtain a result is shortened (Neil et al., 2021). However, the authors did not demonstrate the applicability of their method to the direct detection of PBP2a in blood culture samples without a prior subculture step.

[0034] To date, no technology meeting all the criteria required for implementation in a hospital setting, including short analysis time, simplicity of sample preparation, limited cost and satisfactory performance, is known.

[0035] In order to meet the current needs of analytical laboratories, a method for identifying MRS A strains should meet at least the following criteria: - No subculture step for bacteria isolated from blood (analysis directly from the blood culture bottle when it is detected as positive), - Short sample preparation and analysis time (less than 2 hours) to obtain a result quickly, thus allowing the patient to receive appropriate antibiotic treatment as soon as possible. - low cost so that the test can be carried out systematically, - test detecting both PBP2a and PBP2c proteins with a sensitivity and specificity close to 100%, including variant proteins with a peptide sequence having sequence identity with SEQ ID NO. 1 or 2 sequences.

[0036] The inventors have developed a targeted method for detecting MRSA strains by liquid chromatography coupled with mass spectrometry, carried out directly from a positive blood culture bottle, including an induction step and rapid sample preparation, and allowing a result to be obtained in less than 1h30 from the observation of the presence of a Staphylococcus aureus strain in a biological sample. Description of the invention

[0037] The present invention relates to a method for determining the methicillin resistance properties of a strain of Staphylococcus aureus present in a biological sample, comprising the following steps: a) incubation of the biological sample containing said strain of S. aureus for at least 15 minutes, in the presence of a beta-lactam antibiotic chosen from the following group: cefoxitin and 6-APA (6-aminopenicillanic acid), b) isolation of the bacteria present in said biological sample, c) lysis of the bacteria and hydrolysis of the bacterial proteins, in order to obtain a mixture of peptides, d) analysis of this mixture of peptides by targeted mass spectrometry, the detection of at least one peptide from the PBP2a protein (SEQ ID NO. 1) or PBP2c (SEQ ID NO. 2) during this analysis step being indicative of the methicillin resistance of the Staphylococcus aureus strain present in said biological sample.

[0038] The present invention also relates to a kit for implementing this method, comprising: - an antibiotic from the beta-lactam class chosen from the following group: cefoxitin and 6-APA (6-aminopenicillanic acid), - trypsin, - a reagent allowing selective lysis of non-bacterial cells present in the biological sample, in particular a detergent chosen from saponin, Triton X100 and Sodium Dodecyl Sulfate (SDS), - optionally, reagents for performing targeted mass spectrometry. DESCRIPTION OF THE FIGURES

[0039] [Fig. 1] shows a chromatogram resulting from an MRM analysis of the MRSA26b strain. A) Sample prepared without the induction step. B) Sample prepared with the induction step. The peaks corresponding to the PBP2a peptide transitions are indicated by arrows.

[0040] [Fig. 2] shows the chromatograms resulting from an MRM3 analysis of the MRSA26b strain. A1 and A2) MRM3 chromatograms of the peptides VALELGSK (A1) and FQITTSPGSTQKK (A2) for the sample prepared without the induction step. B1 and B2) MRM3 chromatograms of the peptides VALELGSK (B1) and FQITTSPGSTQKK (B2) for the sample prepared with the induction step.

[0041] [Fig. 3] shows a chromatogram resulting from an MRM analysis of the MRSA28b strain. A) Sample prepared without the induction step. B) Sample prepared with the induction step. The peaks corresponding to the PBP2a peptide transitions are indicated by arrows.

[0042] [Fig. 4] shows the chromatograms resulting from an MRM3 analysis of the MRSA28b strain. A1 and A2) MRM3 chromatograms of the peptides VALELGSK (A1) and FQITTSPGSTQKK (A2) for the sample prepared without the induction step. B1 and B2) MRM3 chromatograms of the peptides VALELGSK (B1) and FQITTSPGSTQKK (B2) for the sample prepared with the induction step.

[0043] [Fig. 5] shows chromatograms of the MSSAlôb strain after an induction step. A) MRM chromatograms. B1 and B2) MRM3 chromatograms for the peptides VALELGSK (B1) and FQITTSPGSTQKK (B2). No peptide is detected for either type of analysis.

[0044] [Fig. 6] shows chromatograms of the MSSAlb strain after an induction step. A) MRM chromatograms. B1 and B2) MRM chromatograms for the peptides VALELGSK (B1) and FQITTSPGSTQkk (B2). No peptide is detected for either type of analysis.

[0045] [Fig. 7] shows a chromatogram resulting from an MRM analysis of a strain expressing PBP2c. A) Sample prepared without the induction step. B) Sample prepared with the induction step. The peaks corresponding to the PBP2c peptide transitions are indicated by arrows. DETAILED DESCRIPTION OF THE INVENTION

[0046] The present invention relates to a method for detecting strains of Staphylococcus aureus. methicillin-resistant lococcus aureus, known as MRAS strains, allowing a result to be obtained in less than 1h30 from a biological sample identified as containing a strain of S. aureus.

[0047] Methicillin, also spelled methicillin, is a beta-lactam antibiotic belonging to the penicillin subfamily. Its CAS number is 61-32-5. It was widely used against Staphylococcus aureus infections before being superseded by cloxacillin, which presents less risk of bacterial resistance.

[0048] Advantageously, the process of the invention makes it possible to identify strains resistant to this antibiotic (MRAS) with a sensitivity and specificity close to 100%.

[0049] The method according to the invention uses a biological sample without a bacterial subculture step, for example a positive blood culture sample (containing blood cells and bacteria). A first step of inducing expression of the PBP2a or PBP2c protein, using a beta-lactam antibiotic, is followed by a rapid isolation step of bacteria, then a step of bacterial lysis and enzymatic digestion of the proteins, and finally an analysis by targeted mass spectrometry for the detection of peptides resulting from the enzymatic digestion of the PBP2a or PBP2c protein.

[0050] Thus, the present invention relates to a method for determining the methicillin resistance properties of a strain of Staphylococcus aureus present in a biological sample, comprising the following steps: a) incubation of the biological sample containing said strain of Staphylococcus aureus for at least 15 minutes, in the presence of a beta-lactam antibiotic chosen from the following group: cefoxitin and 6-APA (6-aminopenicillanic acid), b) isolation of the bacteria present in said biological sample, c) lysis of the bacteria and hydrolysis of the bacterial proteins, in order to obtain a mixture of peptides, d) analysis of this mixture of peptides by targeted mass spectrometry, the detection of at least one peptide from the PBP2a or PBP2c protein during this analysis step being indicative of the methicillin resistance of the Staphylococcus aureus strain present in said biological sample.

[0051] This process was developed from the process described in international application WO 2011 / 045544.

[0052] Based on this process, the inventors identified that the PBP2a protein is expressed very heterogeneously among MRSA strains, at very different levels of expression.

[0053] Using a methodology similar to that presented in application WO 2011 / 045544, but with a Staphylococcus aureus positive blood culture medium as the starting biological sample, i.e. without a bacterial subculture step, it was shown on a cohort of 98 MRSA strains representative of the French epidemiology that about 60% of said MRSA strains have a level of PBP2a expression too low to be detected by the methodology described (see experimental part).

[0054] Indeed, heterogeneity in PBP2a expression exists between different MRSA strains. Some strains naturally express PBP2a at high levels (detectable without induction) and others have very low basal expression levels, not allowing detection of PBP2a by direct analysis, without an induction step.

[0055] It was therefore concluded that the technology described in application WO 2011 / 045544 is not suitable for implementation in a clinical analysis laboratory, as many MRSA strains cannot be detected.

[0056] The present application relates to an improvement of said method, consisting of the addition of a step (a) of incubating the biological sample containing said strain of S. aureus for at least 15 minutes, in the presence of a beta-lactam antibiotic, to induce the expression of the PBP2a protein or the PBP2c protein and thus have a new expression sufficient to detect the variant protein expressed in 100% of MRSA strains.

[0057] The PBP2a protein has the following sequence:

[0058] Sequence 1, PBP2a, Staphylococcus aureus (NCBIID: WP_001801873.1):

[0059] MMKKIKIVPLILIVVVVGFGIYFYASKDKEINNTIDAIEDKNFKQVYKDSSYIS KSDNGEVEMTERPIKIYNSLGVKDINIQDRKIKKVSKNKKRVDAQYKIKTNYG NIDRNVQFNFVKEDGMWKLDWDHSVIIPGMQKDQSIHIENLKSERGKILDRNN VELANTGTAYEIGIVPKNVSKKDYKAIAKELSISEDYIKQQMDQNWVQDDTFV PLKTVKKMDEYLSDFAKKFHLTTNETESRNYPLGKATSHLLGYVGPINSEELK QKEYKGYKDDAVIGKKGLEKLYDKKLQHEDGYRVTIVDDNSNTIAHTLIEKK KKDGKDIQLTIDAKVQKSIYNNMKNDYGSGTAIHPQTGELLALVSTPSYDVYP FMYGMSNEEYNKLTEDKPEPLNKFQITTSPGSTQKILTAMIGLNNKTLDDKT SYKIDGKGWQKDKSWGGYNVTRYEVVNGNIDLKQAIESSDNIFFARVALELG SKKFEKGMKKLGVGEDIPSDYPFYNAQISNKNLDNEILLADSGYGQGEILINPV QILSIYSALENNGNINAPHLLKDTKNKVWKKNIISKENINLLTDGMQQVVNKT HKEDIYRSYANLIGKSGTAELKMKQGETGRQIGWFISYDKDNPNMMMAINVK DVQDKGMASYNAKISGKVYDELYENGNKKYDIDE

[0060] The protein PBP2c presents the following sequence:

[0061] Sequence 2, PBP2c, Staphylococcus aureus (NCBI ID : WP_000725529.1) :

[0062] MKKIYISVLVLLLIMIIITWLFKDDDIEKTISSIEKGNYNEVYKNSSEKSKLAYG EEEIVDRNKKIYKDLSVNNLKITNHEIKKTGKDKKQVDVKYNIYTKYGTIRRN TQLNFIYEDKHWKLDWRPDVIVPGLKNGQKINIETLKSERGKIKDRNGIELAKT GNTYEIGIVPNKTPKEKYDDIARDLQIDTKAITNKVNQKWVQPDSFVPIKKINK QDEYIDKLIKSYNLQINTIKSRVYPLNEATVHLLGYVGPINSDELKSKQFRNYS KNTVIGKKGLERLYDKQLQNTDGFKVSIANTYDNKPLDTLLEKKAENGKDLH LTIDARVQESIYKHMKNDDGSGTALQPKTGEILALVSTPSYDVYPFMNGLSNN DYRKLTNNKKEPLLNKFQITTSPGSTQKILTSIIALKENKLDKNTNFDIYGKGW QKDASWGNYNITRFKVVDGNIDLKQAIESSDNIFFARIALALGAKKFEQGMQD LGIGENIPSDYPFYKAQISNSNLKNEILLADSGYGQGEILVNPIQILSIYSALENN GNIQNPHVLRKTKSQIWKKDIIPKKDIDILTNGMERVVNKTHRDDIYKNYARII GKSGTAELKMNQGETGRQIGWFVSYNKNNPNMLMAINVKDVQNKGMASYN ATISGKVYDDLYDNGKTQFDIDQ

[0063] Advantageously, the process of the present invention makes it possible to detect both the proteins PBP2a and PBP2c, but also variant proteins.

[0064] A "variant protein" is defined as a protein having a peptide sequence with a high sequence identity with the SEQ ID NO. 1 or 2 sequences, in particular a sequence identity of at least 90%, or better yet at least 95%, or even 99% with one of the SEQ ID NO. 1 or SEQ ID NO. 2 sequences. Variant proteins generally have one, two or three point mutations in the sequences of the wild-type proteins, that is to say they differ only in one, two or three amino acids in the peptide sequence.

[0065] The identity percentages referred to in the disclosure of the present invention are determined after optimal alignment of the sequences to be compared, which may therefore include one or more additions, deletions, truncations and / or substitutions.

[0066] This percentage of identity can be calculated by any sequence analysis method well known to a person skilled in the art.

[0067] The percentage of identity can be determined after global alignment of the sequences to be compared, taken in their entirety, over their full length. In addition to manual methods, it is possible to determine the global alignment of sequences using the Needleman and Wunsch algorithm (1970).

[0068] For amino acid sequences, sequence comparison can be performed using any software well known to those skilled in the art, such as Needle. The parameters used may include, in particular, the following: "Gap Open" equal to 10.0, "Gap Extend" equal to 0.5 and the BLOSUM62 matrix.

[0069] Preferably, the percentage of identity defined within the scope of the present invention is determined by means of a global alignment of the sequences to be compared over their entire length.

[0070] Step (a) of induction of the expression of PBP2a or PBP2c

[0071] Induction consists of carrying out a rapid incubation in the presence of an antibiotic in order to activate the regulatory systems of the expression of PBP2a or PBP2c proteins and thus lead to an overexpression of these proteins.

[0072] The antibiotic used is from the beta-lactam class, that is to say, an antibiotic containing a beta-lactam nucleus. This antibiotic is chosen from cefoxitin and 6-APA (6-aminopenicillanic acid).

[0073] Cefoxitin, CAS number 35607-66-0, is a beta-lactam antibiotic classified as a second-generation cephalosporin. Cefoxitin exerts a bactericidal effect by inhibiting cell wall synthesis.

[0074] 6-Aminopenicillanic acid, abbreviated 6-APA, CAS number 551-16-6, is a penicillin derivative.

[0075] Preferably, the antibiotic used for the induction step (a) is cefoxitin.

[0076] Induction is carried out by incubating the biological sample in the presence of an appropriate amount of antibiotic, typically at a temperature between 30° and 40° Celsius. The incubation of the biological sample will preferably take place at 37°C, with stirring.

[0077] According to a particular implementation of the process of the invention, the incubation step (a) is carried out for a period of at least 15 minutes.

[0078] Advantageously, the incubation time may be 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes or even 45 minutes.

[0079] As shown in the examples, in the absence of this induction step, some methicillin-resistant S. aureus strains are not identified as such, because the detection of one of the PBP2a or PBP2c proteins is impossible due to the too low expression of said protein in said strains.

[0080] Step (b) of isolating bacteria

[0081] Bacteria can be isolated by centrifugation.

[0082] According to one embodiment of the process, step (b) also includes a step of selective lysis of non-bacterial cells present in the biological sample, this selective lysis step being carried out before centrifugation of the sample.

[0083] Detergent compounds cause lysis of animal cells by dissociating the membrane. Since bacteria are composed of a rigid peptidoglycan cell wall, they are not lysed by the action of the detergent.

[0084] A detergent compound may, for example, be selected from the following group of compounds: saponin, Triton X100 or Sodium Dodecyl Sulfate (SDS)

[0085] In particular, when the biological sample is a blood sample, step (b) The isolation of bacteria is advantageously carried out concurrently with the lysis of blood cells present in the sample, through the addition of a detergent compound.

[0086] Step (c) of bacterial lysis and hydrolysis of bacterial proteins

[0087] Various methods of bacterial cell lysis can be used. Examples include thermal methods (temperature above 100°C for 10 minutes, or freezing in liquid nitrogen), enzymatic methods (action of lysozyme or lyticase) or mechanical methods (high pressure, grinding or sonication).

[0088] According to a preferred embodiment of the process of the invention, the lysis of bacteria is carried out by sonication.

[0089] The hydrolysis of bacterial proteins into peptides is generally carried out using one of the following two processes: a) By the action of chemical agents such as hydroxyl radicals which induce random cleavages at the level of peptide bonds, or b) By the action of proteolytic enzymes (proteases) which have a hydrolysis action on protein bonds.

[0090] According to a preferred embodiment of the process, the hydrolysis of bacterial proteins in step (c) is an enzymatic hydrolysis.

[0091] Among the commonly used proteases, we will mention pepsin which hydrolyzes peptide bonds preferentially before aromatic amino acids (Tyrosine, Tryptophan and Phenylalanine), endoproteinase GluC which cuts the peptide bond at the level of glutamate residues, or trypsin which cleaves proteins on the C-terminal side of the amino acids lysine and arginine.

[0092] According to a preferred embodiment of the process of the invention, trypsin is used as an enzyme for the step of hydrolyzing bacterial proteins. Trypsin is preferred because of the specificity of its activity, the suitable size of the peptides it generates, and the nature of the "tryptic" peptides, which have a positively charged amino acid (lysine or arginine) on the C-terminal end, thus facilitating analysis by mass spectrometry (analysis of charged molecules).

[0093] A specific protocol for step (c) is presented in the experimental part.

[0094] Step (d) of targeted mass spectrometry analysis

[0095] Mass spectrometry is a physical analytical technology used to detect and identify molecules of interest. It is also known as single reaction monitoring, multiple reaction monitoring, or parallel reaction monitoring. Its principle lies in the separation of charged molecules (ions) in the gas phase according to their mass-to-charge ratio (m / z).

[0096] Mass spectrometers include: (i) an ionization source intended to ionize the molecules to be analyzed, i.e. to give these molecules a positive or negative charge; ii) a mass analyzer designed to separate ionized molecules according to their mass-to-charge ratio (m / z); iii) a detector intended to measure the signal produced either directly by molecular ions, or by ions produced from molecular ions, as detailed below.

[0097] The ionization step required for mass spectrometry can be carried out by any method known to those skilled in the art. The ionization source allows the molecules to be measured to be brought into 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 being analyzed.

[0098] The mass analyzer in which the step of separating ionized markers according to their mass / charge ratio (m / z) is implemented is any mass analyzer known to those skilled in the art. Examples include low-resolution analyzers, such as quadrupole (Q) or 3D ion trap (IT) or linear (LIT) type, also called ion traps, and high-resolution analyzers, which allow for the exact measurement of the analytes and which notably use magnetic sectors coupled to electrical sectors, time of flight (TOF), and orbitrap.

[0099] The separation of molecular ions based on their m / z ratio can be performed once (simple 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 performed, the analysis is called MS / MS / MS or MS3.

[0100] Targeted mass spectrometry is a variant in which the molecules of interest sought by this analytical technique are known beforehand, and the analysis serves to identify their presence or absence in a sample.

[0101] Targeted approaches (Multiple Reaction Monitoring, MRM; Parallel Reaction Monitoring, PRM; Multiple Reaction Monitoring-High Resolution, MRM-HR; Multiple Reaction Monitoring cubed, MRM3, DIA / SWATH) consist of selecting, by a first analyzer, a precise mass corresponding to the peptide of interest, which is then fragmented in a collision cell. The generated fragments are then monitored by a third analyzer, and their signal / intensity is measured.

[0102] In the case of chromatographic couplings, the signal of the fragments is measured as a function of time to be represented in the form of a chromatogram, the appearance of concomitant chromatographic peaks (simultaneous detection of fragments) thus being evidence of the presence of the peptide in the sample.

[0103] These approaches require prior work in selecting and validating the marker peptides to ensure their uniqueness of sequence / mass (or mass / charge ratio, m / z) and their detectability.

[0104] The principle of SRM mode, or MRM mode, is to specifically select a precursor ion, fragment it, and then specifically select one of its fragment ions. For such applications, triple quadrupole devices or triple quadrupole hybrid ion-trapping devices are generally used.

[0105] The DIA / SWATH analysis mode consists of recording fragmentation spectra of contiguous selection windows of precursor ions, most often overlapping by 1 unit of mass-to-charge ratio (m / z), the windows being characterized by a fixed or variable width in m / z, or by using a sliding window of fixed width in m / z, while ensuring that the total cycle time to cover all of these windows allows each chromatographic peak to be sampled by at least 5 points.

[0106] According to a preferred embodiment of the invention, mass spectrometry analysis is targeted during the analysis (SRM / MRM, MRM, MRM3, PRM type mode) or after the analysis (DIA / SWATH type mode).

[0107] More preferably, the targeted mass spectrometry analysis is carried out in MRM or MRM3 mode, and most preferably is carried out in MRM3 mode.

[0108] Targeted mass spectrometry is coupled with peptide separation, preferably by chromatographic or electrophoretic separation of peptides.

[0109] The separation of peptides can be carried out by any technique known to those skilled in the art, and in particular can be carried out by reversed-phase liquid chromatography, normal-phase liquid chromatography, hydrophilic-phase liquid chromatography, or capillary electrophoresis.

[0110] Preferably, the separation of peptides is carried out by reversed-phase liquid chromatography.

[0111] The peptides sought during this analysis step are peptides derived from PBP2a or PBP2c proteins, specific to said proteins.

[0112] It is understood that at least one peptide from these proteins may be detected, but that preferably several peptides will be detected during the analysis, which supports the results obtained.

[0113] According to a preferred embodiment of the method of the invention, the at least one peptide from the PBP2a or PBP2c protein that is detected, i.e. whose presence is demonstrated by targeted mass spectrometry, is chosen from the group of 12 peptides having the following sequences: SEQ ID NO. 3 to SEQ ID NO. 14 shown in Tables 1 and 2 below.

[0114] [Tables 1] SEQ ID NO. Peptide from PBP2A Location in the sequence SEQ ID NO.l 3 IYNSLGVK 70-77 4 DINIQDR 78-84 5 ELSISEDYIK 190 - 199 6 FQITTSPGSTQK 396 - 407 7 ILTAMIGLNNK 408 - 418 8 YEVVNGNIDLK 447 - 457 9 VALELGSK 471 -478 10 SYANLIGK 591 - 598

[0115] [Tables2] SEQ ID NO. Peptide from PBP2c Location in the sequence SEQ ID NO.2 11 LAYGEEEIVDR 52-62 12 SYNLQINTIK 227 - 236 13 ILTSIIALK 404 - 412 14 IALALGAK ​​467 - 474

[0116] Advantageously, all these peptides were selected from conserved areas of the proteins and are therefore also identified in variant proteins of PBP2a and PBP2c.

[0117] The process according to the invention can be carried out on any type of biological sample likely to contain a strain of Staphylococcus aureus.

[0118] According to a particular embodiment of the process, the biological sample is chosen from: - A biological fluid, for example blood, serum, plasma, urine, cerebrospinal fluid, and tears; - A bacterial culture, for example a blood culture, a bacterial colony on agar, a bacterial culture broth; - A food sample, and - any other type of biological sample.

[0119] This will preferably be a biological fluid, in particular a blood sample (blood, serum, plasma), and more particularly the medium of a blood culture positive containing Staphylococcus aureus.

[0120] By “blood culture” is meant a blood sample taken from a patient and then incubated under suitable conditions to allow the proliferation of any bacteria present in said sample.

[0121] This blood culture can be carried out in blood culture bottles such as those marketed in the Bact / Alert range distributed by BioMérieux, or those in the Bactec range distributed by Becton Dickinson.

[0122] Kit for implementing the process

[0123] The present invention also relates to a kit for implementing the process as described above, comprising: - an antibiotic from the beta-lactam class chosen from the following group: ce-foxitin and 6-APA (6-aminopenicillanic acid), - trypsin, - a reagent allowing selective lysis of non-bacterial cells present in the biological sample, for example a detergent.

[0124] For example, among the reagents allowing selective lysis of non-bacterial cells, a detergent compound selected from the following group of compounds may be mentioned: saponin, Triton X100 or Sodium Dodecyl Sulfate (SDS). EXAMPLES

[0125] The examples presented below are intended to illustrate the process according to the invention, but are in no way a limitation of the object of the invention.

[0126] In particular, in the examples presented below, the antibiotic used during the induction step is cefoxitin, but it is understood that 6-APA may also be used.

[0127] Example 1. Materials and Methods

[0128] The induction step (a) is carried out according to the following protocol: - Perform aseptic technique on the septum of the blood culture bottle - Using a syringe and a 21G needle, withdraw 3.8 mL of positive blood culture medium and transfer it into a 15 mL tube. - Add 200 pL of a cefoxitin solution at 8 pg / mL - Homogenize the mixture and then incubate the tube at 37°C with shaking (180 rpm) for 30 minutes

[0129] Step (b) of isolating the bacteria is advantageously carried out concurrently with lysis of the blood cells, according to the following protocol: - After the 30 minutes of induction, transfer 1 mL of medium into a 1.5 mL tube and add 200 µl of a 12% SDS solution then vortex for 10 seconds. - Centrifuge for 2 minutes at 16100 g and discard the supernatant - Resuspend the pellet in 1 mL of physiological saline - Centrifuge for 1 minute at 16100 g and discard the supernatant - Resuspend in 1 mL of physiological saline

[0130] Step (c) of mechanical lysis of bacteria and enzymatic digestion of proteins is carried out according to the following protocol: - Transfer 200 pL of the previously prepared bacterial suspension into a 1.5 mL Eppendorf LoBind tube containing approximately 70 mg of glass beads (Glass beads, acid-washed, 150-212 µm, Sigma-Aldrich, ref G1145) - Add 50 µL of a trypsin solution at 1 mg / mL prepared extemporaneously in 150 mM ammonium bicarbonate buffer from lyophilized trypsin - Immediately place the sample in the water bath of a sonicator set to 50°C and then begin 10 ultrasound cycles (low power) • 30 seconds of ultrasound running • 30 seconds of ultrasound stopped - Immediately after the 10 ultrasound cycles, add 5 pL of formic acid. - Centrifuge the tube at 9600 g for 5 minutes. - Transfer 150 pL of supernatant into a 2 mL amber glass bottle fitted with an insert for mass spectrometry analysis.

[0131] Step (d) of targeted mass spectrometry analysis is carried out according to the following protocol:

[0132] A 5 pL volume of sample from the previous lysis / digestion step is injected into the chromatographic system. The analysis is performed on a Waters XBridge Peptide BEH Cl8 reversed-phase column, 1 mm internal diameter, 100 mm length, 3.5 µm particle size, 130 Å pore size, using a chromatographic system equipped with an Agilent 1290 infinity LC pump, an Agilent 1290 Auto-tosampler autosampler, and an Agilent 1290 TCC column oven set at 60 °C. The gradient used for the chromatographic separation is shown in Table 3.

[0133] Solvent A: H2O + 0.1% formic acid

[0134] Solvent B: Acetonitrile + 0.1% formic acid Time (min) Flow rate (pL / min) Solvent A (%) Solvent B (%) 0 75 98 2 0.5 75 90 10 15.5 75 60 40 15.7 150 25 75 17 150 25 75 17.1 150 98 2 21.2 150 98 2 21.3 75 98 2 22 75 98 2

[0136] The output of the chromatographic system is directly connected to the ionization source of a QTRAP 6500+ mass spectrometer (Sciex) for online analysis of peptides resulting from bacterial protein digestion. The mass spectrometer is operated in MRM or MRM-cubed (MRM3) mode, and the transitions observed for the PBP2a protein are detailed in Table 4. Numér o de la transit! on m / z filter in Q1 m / z filter in Q3 Due 11 time (ms) Peptide Etat the charge of precurse or Ion fragment (gé) Potentiel d’orifice (Declustering potential) (V) Energy and collisi on (V) 1 437.2 25 645.33 1 20 DINIQDR 2 y5 43 24.6 2 437.2 25 531.28 9 20 DINIQDR 2 y4 43 24.6 3 437.2 25 418.20 4 20 DINIQDR 2 y3 43 24.6 4 594.3 44 961.51 4 20 ILTAMIGL NNK 2 y9 47.3 30.3 5 594.3 44 860.46 6 20 ILTAMIGL NNK 2 y8 47.3 30.3 6 594.3 44 789.42 9 20 ILTAMIGL NNK 2 y7 47.3 30.3 7 594.3 44 658.38 8 20 ILTAMIGL NNK 2 y6 47.3 30.3 8 594.3 44 545.30 4 20 ILTAMIGL NNK 2 y5 47.3 30.3 9 598.8 06 954.47 8 20 ELSISEDYI K 2 y8 47.5 30.4 10 598.8 06 867,44 6 20 ELSISEDYI K 2 y7 47,5 30,4 11 598,8 06 754,36 2 20 ELSISEDYI K 2 y6 47,5 30,4 12 598,8 06 667,33 20 ELSISEDYI K 2 y5 47,5 30,4 13 647,8 36 1019,5 37 20 FQITTSPG STQK 2 ylO 90 31 14 647,8 906,45 20 FQITTSPG 2 y9 90 31 36 3 STQK 15 647.8 36 805.40 5 20 FQITTSPG STQK 2 y8 90 31 16 647.8 36 704.35 7 20 FQITTSPG STQK 2 y7 90 31 17 647.8 36 617.32 5 20 FQITTSPG STQK 2 y6 90 31 18 447.2 58 780.42 5 20 IYNSLGVK 2 y7 43.3 25 19 447.2 58 617.36 2 20 IYNSLGVK 2 y6 43.3 25 20 447.2 58 503.31 9 20 IYNSLGVK 2 y5 43.3 25 21 447.2 58 416.28 7 20 IYNSLGVK 2 y4 43.3 25 22 433.2 43 778.44 6 20 SYANLIGK 2 y7 42.9 24.5 23 433.2 43 615.38 2 20 SYANLIGK 2 y6 42.9 24.5 24 433.2 43 544.34 5 20 SYANLIGK 2 y5 42.9 24.5 25 408.7 45 717.41 4 20 VALELGS K 2 y7 20 20 26 408.7 45 646.37 7 20 VALELGS K 2 y6 20 20 27 408.7 45 533.29 3 20 VALELGS K 2 y5 20 20 28 408.7 45 404.25 20 VALELGS K 2 y4 20 20 29 632.3 33 971.55 2 20 YEVVNGN IDLK 2 y9 48.4 31.6 30 632.3 33 872.48 4 20 YEVVNGN IDLK 2 y8 48.4 31.6 31 632.3 773.41 20 YEVVNGN 2 y7 48.4 31.6 33 5 IDLK 32 632.3 33 659.37 2 20 YEVVNGN IDLK 2 y6 48.4 31.6

[0138]

[0139] Table 4. List of transitions followed for the detection of PBP2a. The transitions followed for the PBP2c protein are detailed in Table 5. Transition number m / z filtered in Q1 m / z filtered in Q3 Dwe 11 time (ms) Peptide Precursor charge state Fragment ion (single-charged) Declustering potential (V) Collision energy (V) 43 647.32 0 946.44 8 20 LAYGEE EIVDR 2 y8 48.8 32.2 44 647.32 0 889.42 6 20 LAYGEE EIVDR 2 y7 48.8 32.2 45 647.32 0 760.38 4 20 LAYGEE EIVDR 2 y6 48.8 32.2 46 647.32 0 631.34 1 20 LAYGEE EIVDR 2 y5 48.8 32.2 47,647.32 0 502.29 8 20 LAYGEE EIVDR 2 y4 48.8 32.2 48,597.33 0 943.55 7 20 SYNLQIN TIK 2 y8 47.4 30.4 49 597.33 0 829.51 4 20 SYNLQIN TIK 2 y7 47.4 30.4 50 597.33 0 716.43 0 20 SYNLQIN TIK 2 y6 47.4 30.4 51 597.33 0 588.37 2 20 SYNLQIN TIK 2 y5 47.4 30.4 52 597.33 0 475.28 7 20 SYNLQIN TIK 2 y4 47.4 30.4 53 597.33 0 361.24 5 20 SYNLQIN TIK 2 y3 47.4 30.4 54 486.32 9 745.48 2 20 ILTSIIAL K 2 y7 44.4 26.4 55,486.32 9,644.43 4 20 ILTSIIAL K 2 y6 44.4 26.4 56,486.32 557.40 20 ILTSIIAL 2 y5 44.4 26.4 9 2 K 57,486.32 9,444.31 8 20 ILTSIIAL K 2 y4 44.4 26.4 58,486.32 9,331.23 4 20 ILTSIIAL K 2 y3 44.4 26.4 59,378.75 3,643.41 4 20 IALALG AK 2 y7 41.5 22.5 60 378.75 3 572.37 7 20 IALALG AK 2 y6 41.5 22.5 61 378.75 3 459.29 3 20 IALALG AK 2 y5 41.5 22.5 62 378.75 3,388.25 5 20 IALALG AK 2 y4 41.5 22.5 63,378.75 3,275.17 1 20 IALALG AK 2 y3 41.5 22.5 64,378.75 3,218.15 0 20 IALALG AK 2 y2 41.5 22.5

[0141] The mass spectrometer parameters for analysis in MRM mode are detailed in Table 6 below. MRM Scan Type, Positive Polarity, Turbo Spray IonDrive Ionization Source (SCIEX), Q1 Unit Resolution, Q3 Unit Resolution, Inter-scan Pause 5 ms, Scan Speed ​​10 Da / sec, Curtain Gas 50.00 psi, Cone Voltage 5500.00 V, Source Temperature 550.00 °C, Nebulizing Gas (GSI) 70.00 psi, Heating Gas (GS2) 60.00 psi, High Collision Gas, Input Potential (EP) 10.00 V, Collision Cell Output Potential (CXP) 12.00 V, Analyst Software Version 1.7.2

[0143] The mass spectrometer parameters for analysis in MRM3 mode are detailed in Table 7 (peptide VALELGSK) and in Table 8 (peptide FQITTSPGSTQK) below. Scanning Type MS / MS / MS (MS3) First Precursor 408.75 Da Second Precursor 646.38 Da Centered Analysis (3rd Generation Ion) 404.25 Da Scanning Window 2 Da Polarity Positive Ionization Source Turbo Spray lonDrive (SCIEX) Q1 Resolution unit Q3 Resolution LIT Fixed Linear Ion Trap Fill Time 200 ms Excitation Time 25 ms Q3 Entry Barrier 8 V Inter-scan Pause 15 ms Q0 Trapping Enabled Scanning Speed ​​1000 Da / sec Curtain Gas 50.00 psi Cone Voltage 5500.00 V Source Temperature 550.00 °C Nebulizing Gas (GSI) 70.00 psi Heating Gas (GS2) 60.00 psi High Collision Gas Orifice Potential (DP) 20 V Collision Energy (CE) 20 V Collision Energy Propagation (CES) OV Excitation Energy (AF2) 0.1 V Input Potential (EP) 10.00 V Collision Cell Output Potential (CXP) 12.00 V Analyst Software Version 1.7.2 Scanning Type MS / MS / MS (MS3) First Precursor 647.84 Da Second Precursor 805.41 Da Centered Analysis (3rd Generation Ion) 617.33 Da Scanning Window 2 Da Polarity Positive Ionization Source Turbo Spray lonDrive (SCIEX) Q1 Resolution unit Q3 Resolution LIT Fixed Linear Ion Trap Fill Time 200 ms Excitation Time 25 ms Q3 Entry Barrier 8 V Inter-scan Pause 15 ms Q0 Trapping Enabled Scanning Speed ​​1000 Da / sec Curtain Gas 50.00 psi Cone Voltage 5500.00 V Source Temperature 550.00 °C Nebulizing Gas (GSI) 70.00 psi Heating Gas (GS2) 60.00 psi High Collision Gas Orbit Potential (DP) 90 V Collision Energy (CE) 31 Collision Energy Propagation (CES) V 0 V Excitation Energy (AF2) 0.11 V Input Potential (EP) 10.00 V Collision Cell Output Potential (CXP) 12.00 V Analyst Software Version 1.7.2

[0146] Example 2. Initial results

[0147] Figures 1 and 2 illustrate the necessity of the induction step for the detection of peptides from PBP2a by MRM.

[0148] Chromatograms A are obtained from the analysis of strains without an induction step, chromatograms B correspond to the analysis of the same strains after an induction step.

[0149] In the absence of induction, the peaks of peptides from PBP2a are confused with background noise and therefore undetectable.

[0150] On the contrary, after an induction step, the characteristic peaks are observed: they are correctly drawn and above the background noise. PBP2a is correctly detected.

[0151] Blood cultures were inoculated for 98 strains of MRSA and 19 strains of methicillin-sensitive Staphylococcus aureus (MSSA) not expressing PBP2a, then the samples were prepared to vial positivity and analyzed according to the protocols of Example 1.

[0152] The area under the curve was measured for each transition. Although not expressing PBP2a, the area under the curve was also measured for samples of MSSA-sensitive strains over the peptide elution window to obtain a baseline value. Indeed, the signal of a transition can be contaminated by noise or interference from the matrix; therefore, it is necessary to evaluate this baseline in a matrix not containing the analyte (here, PBP2a) in order to establish thresholds above which we can confirm the presence of the peptides.

[0153] For each transition, the threshold corresponds to 150% of the maximum area measured on the peptide elution window for the MSSA control samples.

[0154] Example 3. MRM spectrometry results

[0155] All MRM transitions with an area greater than the threshold value were considered positive and are marked with a 1 in the following results tables. Transitions with an area less than the threshold value were marked with a 0 and therefore considered negative. A sample was considered identified as containing an MRSA strain if at least three peptides were detected with at least two positive transitions.

[0156] Tables 9 and 10 below correspond to the results obtained during the MRM analysis of the 98 MRSA without the induction step.

[0157] Tables 11 and 12 below correspond to the results obtained during the MRM analysis of the 98 MRSA with the induction step. Transition Number 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2 2 3 2 4 2 5 2 6 2 7 2 8 2 9 3 0 3 1 3 2 MRS Alb 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 0 0 0 1 0 0 0 0 0 1 1 1 1 MRS A2b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 1 1 1 1 1 1 1 1 MRS A3b 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 MRS A4b 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 MRS A5b 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 MRS A6b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 1 1 1 0 0 1 1 1 1 MRS A7b 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 MRS A8b 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 MRS A9b 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 MRS AlOb 0 0 0 0 0 1 0 0 1 0 1 0 0 0 0 0 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 MRS Allb 0 0 0 0 0 0 0 0 1 1 1 0 1 1 0 0 0 0 1 0 0 0 1 0 1 1 1 1 1 0 1 0 MRS A12b 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0MRS A13b 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 MRS A14b 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 MRS 0 0 0 0 0 0 0 0 1 1 1 1 0 1 0 0 0 0 1 0 0 0 1 0 0 0 0 0 1 0 0 0 A15b MRS A16b 0 0 0 0 0 1 0 0 1 0 1 1 1 1 0 0 0 0 1 0 0 0 1 0 0 0 0 0 1 1 1 0 MRS A17b 0 0 0 0 0 0 0 0 1 0 1 0 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1 1 0 0 MRS A18b 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 1 0 0 0 1 0 0 0 0 0 1 0 0 0 MRS A19b 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 MRS A20b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 0 0 0 0 1 1 1 1 1 MRS A21b 0 0 0 0 0 0 1 1 1 1 1 0 1 1 0 1 0 0 1 0 0 0 1 0 0 0 0 0 1 0 0 0 MRS A22b 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 0 0 0 0 0 MRS A23b 0 0 0 0 0 0 0 0 1 0 1 0 0 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 MRS A24b 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 MRS A25b 0 0 0 0 0 1 0 0 1 1 1 0 1 0 0 0 0 0 1 0 0 0 1 0 0 0 0 0 0 0 1 0 MRS A26b 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 MRS A27b 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 MRS A28b 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 MRS A29b 0 0 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 0 00 0 0 0 0 0 0 0 0 0 0 0 0 MRS A30b 0 0 0 0 0 0 0 0 1 0 1 0 1 1 0 0 0 0 1 0 0 0 1 0 0 0 0 0 1 0 1 0 MRS A31b 0 0 0 0 0 1 1 0 0 0 1 0 0 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1 0 0 0 MRS 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 0 0 0 1 0 0 0 0 0 1 1 1 1 A32b MRS A33b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 0 0 0 1 0 1 1 1 1 1 1 1 1 MRS A34b 0 0 0 0 0 0 0 1 0 0 1 0 1 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 1 0 0 0 MRS A35b 0 0 0 0 0 0 0 0 1 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 MRS A36b 1 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 0 0 0 1 0 0 0 0 0 1 1 1 0 MRS A37b 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 MRS A38b 0 0 0 1 1 1 1 1 1 0 1 0 1 0 0 0 0 0 1 0 0 0 1 0 0 0 0 0 1 0 1 0 MRS A39b 0 0 0 1 1 1 1 1 0 1 1 0 0 1 0 0 1 0 1 0 0 0 1 0 0 0 0 0 0 0 1 0 MRS A40b 0 0 0 1 1 1 1 1 1 0 1 0 1 0 1 0 1 0 1 0 0 0 1 0 0 0 0 0 0 0 1 0 MRS A41b 1 0 0 1 1 1 1 1 1 0 1 0 1 1 1 1 1 0 1 0 0 0 1 0 1 1 1 1 1 1 1 0 MRS A42b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 0 0 0 1 0 1 1 0 1 1 1 1 1 MRS A43b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 1 0 0 0 0 1 1 1 1 MRS A44b 0 0 0 1 1 1 1 1 1 1 1 1 0 0 0 0 1 0 1 0 0 0 1 0 0 0 0 0 0 0 1 1 MRS A45b 1 0 0 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 0 0 0 1 0 0 0 0 0 0 0 1 0 MRS A46b 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 00 0 0 0 0 0 0 0 0 0 0 0 0 MRS A47b 0 0 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 MRS A48b 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 MRS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 A49b

[0159] Table 9: Validation table of MRM transitions from PBP2a of MRSAlb to MRSA49b strains prepared WITHOUT induction step

[0160] [TableauxlO] Numéro de la transition 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2 2 3 2 4 2 5 2 6 2 7 2 8 2 9 3 0 3 1 3 2 MRS A50b 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 MRS A51b 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 MRS A52b 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 MRS A53b 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 MRS A54b 0 0 0 0 0 0 1 0 1 0 1 0 1 1 0 0 1 0 1 0 0 0 1 0 0 0 0 0 1 0 1 0 MRS A55b 0 0 0 0 0 1 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 MRS A56b 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 MRS A57b 0 0 0 0 0 1 0 0 1 0 1 1 1 1 0 0 0 0 1 0 0 0 1 0 0 0 0 0 1 0 0 0 MRS A58b 1 0 0 0 0 1 1 1 1 0 1 1 1 1 1 0 0 0 1 0 0 0 1 0 1 0 0 0 1 1 1 1 MRS A59b 1 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 0 0 0 1 0 0 0 0 0 1 1 1 1 MRS A60b 0 0 0 0 0 1 1 0 1 0 1 0 1 0 0 0 0 0 1 0 0 0 0 0 1 1 1 1 1 0 0 0 MRS A61b 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 00 0 0 0 0 MRS A62b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 0 0 0 0 1 1 1 1 MRS A63b 0 0 0 0 0 0 0 0 0 0 1 0 1 1 0 0 0 0 1 0 0 0 1 0 0 0 0 0 1 1 1 0 MRS 0 0 0 0 0 0 0 1 0 0 1 0 0 0 0 0 0 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 A64b MRS A65b 0 0 0 0 0 0 1 0 1 0 1 1 1 1 0 0 0 0 1 0 0 0 1 0 0 0 0 0 1 1 1 0 MRS A66b 0 0 0 0 0 0 1 0 1 0 1 0 1 1 0 1 0 0 1 0 0 0 1 0 0 0 0 0 1 1 0 0 MRS A67b 0 0 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 MRS A68b 1 1 1 0 0 0 0 0 1 0 1 1 0 1 0 0 1 0 1 0 0 0 1 0 0 0 0 0 1 0 0 0 MRS A69b 0 0 0 0 0 1 0 0 0 0 1 0 1 0 0 0 0 0 1 0 0 0 0 0 1 1 0 1 0 0 0 0 MRS A70b 0 0 0 0 0 0 0 0 1 0 1 0 1 0 0 0 0 0 1 0 0 0 1 0 1 1 0 1 0 0 0 0 MRS A71b 1 1 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 0 1 0 0 0 1 0 0 0 0 0 1 1 1 1 MRS A72b 1 1 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 0 1 0 0 0 1 1 1 1 0 1 1 1 1 1 MRS A73b 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 MRS A74b 1 0 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 0 1 0 0 0 1 0 1 1 1 1 1 1 1 1 MRS A75b 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 MRS A76b 1 1 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 0 1 1 0 1 1 1 1 1 MRS A77b 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 MRS A78b 1 0 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 0 10 0 0 1 0 1 1 1 1 1 1 1 1 MRS A79b 0 0 0 0 0 0 0 0 1 1 1 0 1 1 0 0 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 MRS A80b 1 0 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 0 1 0 0 0 1 0 0 0 0 0 1 1 1 1 MRS 0 0 0 0 0 0 0 0 1 0 1 0 0 1 0 0 0 0 1 0 0 0 1 0 0 0 0 0 1 0 0 0 A81b MRS A82b 1 0 0 0 0 0 0 0 1 0 1 0 1 0 0 1 0 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 MRS A83b 0 0 0 0 0 0 0 0 1 0 1 0 0 1 0 0 0 0 1 0 0 0 1 0 0 0 0 0 1 0 1 0 MRS A84b 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 MRS A85b 0 0 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 MRS A86b 1 0 1 0 0 0 0 0 1 1 1 0 1 1 1 1 1 0 1 0 0 0 1 0 0 0 0 0 1 0 1 1 MRS A87b 0 0 0 0 0 0 0 0 1 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 MRS A88b 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 MRS A89b 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 0 1 0 0 0 1 0 1 1 1 1 0 0 0 0 MRS A90b 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 0 0 0 1 0 1 0 0 1 0 0 0 0 MRS A91b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 1 0 0 0 0 1 1 1 1 MRS A92b 1 0 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 0 1 0 0 0 1 0 0 0 0 1 1 1 1 0 MRS A93b 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 MRS A94b 1 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 0 1 0 0 0 1 0 0 0 0 0 1 1 1 1 MRS A95b 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 10 0 0 1 0 1 1 0 0 1 1 1 1 MRS A96b 0 0 0 0 0 0 0 0 1 1 1 0 0 1 0 0 1 0 1 0 0 0 1 0 0 0 0 0 1 0 0 0 MRS A97b 0 0 0 0 0 0 0 0 1 1 1 1 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 1 0 0 0 MRS 0 0 0 0 0 0 0 0 1 1 1 0 1 1 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 A98b

[0161] Table 10. Validation table of MRM transitions of PBP2a from MRSA50b to MRSA98b strains prepared WITHOUT induction step

[0162] [Tables 11] Transition Number 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2 2 3 2 4 2 5 2 6 2 7 2 8 2 9 3 0 3 1 3 2 MRS Alb 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 0 1 1 1 1 1 1 1 1 1 MRS A2b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 MRS A3b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 0 0 0 1 0 1 1 0 1 1 1 1 1 MRS A4b 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 0 0 0 1 0 1 1 0 1 1 1 1 1 MRS A5b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 0 1 1 1 0 1 1 1 1 MRS A6b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 MRS A7b 1 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 0 0 0 1 0 1 1 0 0 1 1 1 1 MRS A8b 1 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 0 0 0 1 0 1 0 0 0 1 1 1 1 MRS A9b 0 0 0 0 0 0 0 0 1 0 1 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 MRS AlOb 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 0 0 0 1 0 1 1 1 1 1 1 1 1 MRS Allb 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 0 1 0 1 1 1 1 1 1 1 1 MRS A12b 1 0 1 1 0 1 1 1 1 1 1 1 1 1 1 1 0 0 1 0 0 0 1 0 0 0 0 0 1 1 1 1MRS A13b 1 0 0 1 0 0 1 1 1 0 1 0 1 1 1 0 1 0 1 0 0 0 1 0 0 0 0 0 1 0 1 1 MRS A14b 1 0 0 1 0 1 1 0 1 1 1 0 1 1 0 1 0 0 1 0 0 0 1 0 0 0 0 0 1 0 1 0 MRS 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 A15b MRS A16b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 0 1 1 1 1 1 1 1 1 MRS A17b 1 0 0 0 0 1 1 0 1 1 1 1 1 1 0 0 1 0 1 0 0 0 1 0 0 0 0 0 1 1 1 0 MRS A18b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 0 1 1 1 1 1 1 1 1 MRS A19b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 0 0 0 1 0 1 1 0 0 1 1 1 1 MRS A20b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 0 1 1 1 1 1 1 1 1 MRS A21b 0 0 0 0 0 1 1 0 1 0 1 1 1 1 0 1 0 0 1 0 0 0 1 0 0 0 0 0 1 0 1 0 MRS A22b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 0 0 0 1 0 1 1 0 0 1 1 1 1 MRS A23b 1 1 0 1 1 1 1 0 1 0 1 0 1 1 0 1 0 0 1 0 0 0 1 0 0 0 0 0 1 1 1 0 MRS A24b 0 0 0 0 0 1 1 0 1 0 1 0 0 1 0 0 0 0 1 0 0 0 1 0 0 0 0 0 1 1 1 0 MRS A25b 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 0 0 0 1 0 1 1 0 1 1 1 1 1 MRS A26b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 0 1 1 1 1 1 1 1 1 MRS A27b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 0 1 1 0 1 1 1 1 1 MRS A28b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 0 1 1 1 0 1 1 1 1 MRS A29b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 10 0 0 1 0 1 1 0 0 1 1 1 1 MRS A30b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 0 1 1 0 0 1 1 1 1 MRS A31b 1 0 0 1 0 0 0 1 1 0 1 1 1 1 0 1 0 0 1 0 0 0 1 0 0 0 0 0 0 0 1 0 MRS 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 A32b MRS A33b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 MRS A34b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 0 1 1 0 1 1 1 1 1 MRS A35b 1 1 0 0 0 0 1 0 1 0 1 0 1 1 1 1 0 0 1 0 0 0 1 0 0 0 0 0 1 0 1 0 MRS A36b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 0 1 1 1 1 1 1 1 1 MRS A37b 1 0 1 1 0 1 1 1 1 1 1 1 1 1 0 1 0 0 1 0 0 0 1 0 0 0 0 0 1 1 1 0 MRS A38b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 0 1 1 1 1 1 1 1 1 MRS A39b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 MRS A40b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 0 1 1 1 1 1 1 1 1 MRS A41b 0 0 0 1 0 1 1 1 1 0 1 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 MRS A42b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 MRS A43b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 1 1 1 1 1 1 1 1 1 MRS A44b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 0 1 1 1 1 1 1 1 1 MRS A45b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 0 1 1 1 1 1 1 1 1 MRS A46b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10 0 0 1 0 1 1 1 0 1 1 1 1 MRS A47b 1 0 0 1 0 1 1 1 1 0 1 1 1 1 0 0 0 0 1 0 0 0 1 0 0 0 0 0 1 1 1 0 MRS A48b 1 0 0 1 1 1 1 1 1 1 1 1 1 1 0 1 0 0 1 0 0 0 1 0 0 0 0 0 1 1 1 0 MRS 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 0 0 0 1 0 1 1 0 1 1 1 1 1 A49b

[0163] Table 11. Validation table of MRM transitions from PBP2a of MRSAlb to MRSA49b strains prepared WITH induction step

[0164] [Tables 12] Numéro de la transition 1 2 3 4 5 6 7 8 9 1 0 1 1 1 2 1 3 1 4 1 5 1 6 1 7 1 8 1 9 2 0 2 1 2 2 2 3 2 4 2 5 2 6 2 7 2 8 2 9 3 0 3 1 3 2 MRS A50b 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 0 1 1 0 0 1 1 1 1 MRS A51b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 0 1 1 1 1 1 1 1 1 MRS A52b 0 0 0 0 0 1 1 0 1 0 1 0 1 1 1 1 0 0 1 0 0 0 1 0 0 0 0 0 1 0 1 0 MRS A53b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 0 1 1 0 0 1 1 1 1 MRS A54b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 0 1 1 1 1 1 1 1 1 MRS A55b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 0 1 1 1 1 1 1 1 1 MRS A56b 1 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 0 0 0 1 0 1 1 0 0 1 1 1 1 MRS A57b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 0 1 1 1 1 1 1 1 1 MRS A58b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 0 1 1 0 1 1 1 1 1 MRS A59b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 1 1 1 1 1 1 1 1 1 MRS A60b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 0 0 0 1 0 1 1 0 0 1 1 1 1 MRS A61b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 1 1 1 11 1 1 1 1 MRS A62b 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 1 1 1 1 1 1 1 1 1 MRS A63b 1 1 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 0 1 1 0 1 1 1 1 1 MRS 1 1 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 0 1 1 1 1 1 1 1 1 A64b MRS A65b 1 1 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 0 1 1 1 1 1 1 1 1 MRS A66b 1 1 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 0 1 1 1 1 1 1 1 1 MRS A67b 1 1 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 0 1 1 1 1 1 1 1 1 MRS A68b 1 1 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 1 1 1 1 1 1 1 1 1 MRS A69b 1 1 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 0 1 1 1 1 1 1 1 1 MRS A70b 1 1 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 1 1 1 1 1 1 1 1 1 MRS A71b 1 1 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 0 1 1 1 1 1 1 1 1 MRS A72b 1 1 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 MRS A73b 1 0 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 0 1 0 0 0 1 0 0 0 0 0 1 1 1 0 MRS A74b 1 1 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 0 1 1 1 1 1 1 1 1 MRS A75b 1 1 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 0 1 0 0 0 1 0 0 0 0 0 1 1 1 1 MRS A76b 1 1 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 MRS A77b 1 1 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 0 1 1 1 1 1 1 1 1 MRS A78b 1 1 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 11 1 0 1 0 1 1 1 1 1 1 1 1 MRS A79b 1 1 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 0 1 1 1 1 1 1 1 1 MRS A80b 1 1 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 0 1 0 0 0 1 0 1 1 0 1 1 1 1 1 MRS 1 0 0 0 0 0 0 0 1 0 1 0 1 1 0 0 0 0 1 0 0 0 1 0 0 0 0 0 1 1 1 0 A81b MRS A82b 1 1 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 0 1 1 0 1 1 1 1 1 MRS A83b 1 1 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 0 1 0 0 0 1 0 1 1 0 0 1 1 1 1 MRS A84b 1 1 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 0 1 0 0 0 1 0 1 1 0 0 1 1 1 1 MRS A85b 1 1 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 0 1 0 0 0 1 0 0 0 0 0 1 1 1 0 MRS A86b 1 1 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 0 1 1 1 1 1 1 1 1 MRS A87b 1 1 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 1 1 1 1 1 1 1 1 1 MRS A88b 1 1 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 0 1 1 0 0 1 1 1 1 MRS A89b 1 1 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 0 1 1 0 1 1 1 1 1 MRS A90b 1 1 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 0 1 1 1 1 1 1 1 1 MRS A91b 1 1 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 1 1 1 1 1 1 1 1 1 MRS A92b 1 1 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 MRS A93b 1 1 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 0 1 1 1 1 1 1 1 1 MRS A94b 1 1 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 0 1 1 1 1 1 1 1 1 MRS A95b 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 11 0 0 1 1 1 1 1 1 1 1 1 1 MRS A96b 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 0 1 1 1 0 1 1 1 1 MRS A97b 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 0 1 1 0 1 1 1 1 1 MRS 0 0 0 1 0 1 1 1 1 1 1 1 1 1 1 1 1 0 1 0 0 0 1 0 1 1 0 1 1 1 1 0 A98b

[0165] Table 12. Validation table of MRM transitions of PBP2a from MRSA50b to MRSA98b strains prepared WITH induction step

[0166] In view of the results presented in Tables 9 to 12, the following conclusions can be stated:

[0167] Without induction step. 38 out of 98 MRSAs have at least 2 positive transitions for at least 3 peptides, which corresponds to a detection sensitivity of 39%.

[0168] With the induction step, 96 out of 98 MRSAs possess at least 2 positive transitions for at least 3 peptides. This corresponds to a 98% sensitivity for MRSA detection with these validation criteria. Only the MRSA9b and MRSA41b strains are not correctly identified because only 2 peptides are detected with 2 transitions for the MRSA41b strain and 1 peptide with 2 transitions for the MRSA9b strain.

[0169] All MSSAs analyzed using the same method have, for each transition, an area under the curve value lower than the previously established threshold values. This means that no MSSA is identified as an MRSA, which is equivalent to a specificity of 100%.

[0170] Example 4. MRM3 spectrometry results for PBP2A

[0171] MRM3 transitions with an area greater than the threshold value were considered positive and are denoted 1. Transitions with an area less than or equal to the threshold value were denoted 0 and considered negative. A sample was considered identified as MRSA if at least one MRM3 transition was detected as positive. The 19 MSSA strains were analyzed using the same procedure, and the results are presented in the following tables.

[0172] Table 13 shows the results obtained during the MRM3 analysis of the 98 MRSAs without the induction step. It represents the validations of the MRM3 transitions of the 98 MRSAs prepared WITHOUT the induction step.

[0173] Table 14 shows the results obtained during the MRM3 analysis of the 98 MRSAs with the induction step. It represents the validations of the MRM3 transitions of the 98 MRSAs prepared with the induction step.

[0174] [Tables 13] VALELGSK FQITTSPGST QK VALELGSK FQITTSPGST QK 408,75 / 646, 38 / 404,25 647,84 / 805, 41 / 617,33 408,75 / 646, 38 / 404,25 647,84 / 805, 41 / 617,33 MRSAlb 1 1 MRSA50 b 1 1 MRSA2b 1 1 MRSA51 b 1 0 MRSA3b 1 0 MRSA52 b 0 0 MRSA4b 1 1 MRSA53 b 1 1 MRSA5b 1 0 MRSA54 b 1 1 MRSA6b 1 1 MRSA55 b 1 0 MRSA7b 1 0 MRSA56 b 0 0 MRSA8b 0 0 MRSA57 b 1 1 MRSA9b 0 0 MRSA58 b 1 1 MRSA10 b 1 1 MRSA59 b 1 1 MRSA11 b 1 0 MRSA60 b 1 1 MRSA12 b 1 0 MRSA61 b 1 1 MRSA13 b 0 0 MRSA62 b 1 1 MRSA14 b 1 1 MRSA63 b 1 1 MRSA15 1 1 MRSA64 1 1 b b MRSA16 b 1 1 MRSA65 b 1 1 MRSA17 b 1 0 MRSA66 b 1 1 MRSA18 b 1 0 MRSA67 b 1 1 MRSA19 b 0 0 MRSA68 b 1 1 MRSA20 b 1 1 MRSA69 b 1 1 MRSA21 b 1 1 MRSA70 b 1 0 MRSA22 b 1 0 MRSA71 b 1 1 MRSA23 b 1 0 MRSA72 b 1 1 MRSA24 b 0 0 MRSA73 b 0 0 MRSA25 b 1 1 MRSA74 b 1 1 MRSA26 b 0 0 MRSA75 b 1 0 MRSA27 b 1 0 MRSA76 b 1 1 MRSA28 b 1 1 MRSA77 b 1 0 MRSA29 b 1 0 MRSA78 b 1 1 MRSA30 b 1 0 MRSA79 b 1 1 MRS A31 b 1 1 MRSA80 b 1 1 MRSA32 1 1 MRSA81 1 1 b b MRSA33 b 1 1 MRSA82 b 1 1 MRSA34 b 1 1 MRSA83 b 1 1 MRSA35 b 1 0 MRSA84 b 1 0 MRSA36 b 1 1 MRSA85 b 1 1 MRSA37 b 0 0 MRSA86 b 1 1 MRSA38 b 1 1 MRSA87 b 1 1 MRSA39 b 1 1 MRSA88 b 1 0 MRSA40 b 1 1 MRSA89 b 1 1 MRS A41 b 1 1 MRSA90 b 1 1 MRSA42 b 1 1 MRSA91 b 1 1 MRSA43 b 1 1 MRSA92 b 1 1 MRSA44 b 1 1 MRSA93 b 1 0 MRSA45 b 1 1 MRSA94 b 1 1 MRSA46 b 0 0 MRSA95 b 1 1 MRSA47 b 1 0 MRSA96 b 1 0 MRSA48 b 1 0 MRSA97 b 1 0 MRSA49 1 0 MRSA98 1 1 b b

[0175] [Tableaux 14] VALELGSK FQITTSPGST QK VALELGSK FQITTSPGST QK 408,75 / 646, 38 / 404,25 647,84 / 805, 41 / 617,33 408,75 / 646, 38 / 404,25 647,84 / 805, 41 / 617,33 MRSA 1b 1 1 MRSA 50b 1 1 MRSA 2b 1 1 MRSA 51b 1 1 MRSA 3b 1 1 MRSA 52b 1 1 MRSA 4b 1 1 MRSA 53b 1 1 MRSA 5b 1 1 MRSA 54b 1 1 MRSA 6b 1 1 MRSA 55b 1 1 MRSA 7b 1 1 MRSA 56b 1 1 MRSA 8b 1 1 MRSA 57b 1 1 MRSA 9b 1 0 MRSA 58b 1 1 MRSA 10b 1 1 MRSA 59b 1 1 MRSA 11b 1 1 MRSA 60b 1 1 MRSA 12b 1 1 MRSA 61b 1 1 MRSA 13b 1 1 MRSA 62b 1 1 MRSA 14b 1 1 MRSA 63b 1 1 MRSA 1 1 MRSA 1 1 15b 64b MRSA 16b 1 1 MRSA 65b 1 1 MRSA 17b 1 1 MRSA 66b 1 1 MRSA 18b 1 1 MRSA 67b 1 1 MRSA 19b 1 1 MRSA 68b 1 1 MRSA 20b 1 1 MRSA 69b 1 1 MRSA 21b 1 1 MRSA 70b 1 1 MRSA 22b 1 1 MRSA 71b 1 1 MRSA 23b 1 1 MRSA 72b 1 1 MRSA 24b 1 1 MRSA 73b 1 0 MRSA 25b 1 1 MRSA 74b 1 1 MRSA 26b 1 1 MRSA 75b 1 1 MRSA 27b 1 1 MRSA 76b 1 1 MRSA 28b 1 1 MRSA 77b 1 1 MRSA 29b 1 1 MRSA 78b 1 1 MRSA 30b 1 1 MRSA 79b 1 1 MRSA 31b 1 1 MRSA 80b 1 1 MRSA 1 1 MRSA 1 1 32b 81b MRSA 33b 1 1 MRSA 82b 1 1 MRSA 34b 1 1 MRSA 83b 1 1 MRSA 35b 1 0 MRSA 84b 1 1 MRSA 36b 1 1 MRSA 85b 1 1 MRSA 37b 1 1 MRSA 86b 1 1 MRSA 38b 1 1 MRSA 87b 1 1 MRSA 39b 1 1 MRSA 88b 1 1 MRSA 40b 1 1 MRSA 89b 1 1 MRSA 41b 1 1 MRSA 90b 1 1 MRSA 42b 1 1 MRSA 91b 1 1 MRSA 43b 1 1 MRSA 92b 1 1 MRSA 44b 1 1 MRSA 93b 1 1 MRSA 45b 1 1 MRSA 94b 1 1 MRSA 46b 1 1 MRSA 95b 1 1 MRSA 47b 1 1 MRSA 96b 1 1 MRSA 48b 1 1 MRSA 97b 1 1 MRSA 1 1 MRSA 1 1 49b 98b

[0176] 19 strains sensitive to methicillin (MSSAlb, 2b, 3b, 4b, 5b, 6b, 7b, 8b, 9b, 10b, 11b, 12b, 13b, 14b, 15b, 16b, 17b, 18b, and 19b) were tested: no peptides derived from PBP2a were detected, as expected. Results all having a value of zero are not presented in detail.

[0177] Without the induction step. 87 out of 98 strains show at least one positive transition, which corresponds to 89% sensitivity (Table 12).

[0178] With the induction step, all of the MRSA strains tested have at least one positive MRM3 transition, which corresponds to 100% sensitivity (Table 13).

[0179] All MSSAs analyzed using the same method (with the induction step) have, for each transition, an area under the curve value less than or equal to the previously established threshold values. This means that no susceptible MSSA strain is identified as MRSA, which is equivalent to 100% specificity.

[0180] Conclusions

[0181] The performance of the process according to the invention is presented in Table 15 below, which illustrates the importance of the induction step.

[0182] [Tables 15] PBP2a detection sensitivity: Without induction, With induction. Acquisition method: MRM 39%, 98%; MRM3 89%, 100%.

[0183] These results show that the method according to the invention allows rapid identification of MRSA in less than 1h30, directly from a positive blood sample (blood culture), and with performance superior to other methods currently on the market (98% sensitivity and 100% specificity in MRM, and 100% sensitivity and 100% specificity in MRM3).

[0184] The implementation of the sample preparation protocol is simple and the cost of consumables per analysis is minimal.

[0185] Furthermore, the possibility of non-detection, in the case of a variant protein of PBP2a possessing one or more point mutations, is very unlikely, given that the analysis is based on the detection of 8 different peptides of the PBP2a protein.

[0186] Finally, it has also been shown that the induction step of the process is necessary for the detection of the PBP2c protein in a strain of Staphylococcus aureus expressing the protein. The results presented in [Fig. 7] show that detection would have been impossible without the prior induction step by incubation with an anti- biotic of the beta-lactam family. REFERENCES

[0187] WO 2011 / 045544

[0188] Kirby, W. M. M. (1944). Extraction of a highly potent penicillin inactivator from pe-nicillin resistant staphylococci. Science 99, 452-453.

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Claims

Demands

1. A method for determining the methicillin resistance properties of a strain of Staphylococcus aureus present in a biological sample, comprising the following steps: a) incubating the biological sample containing said strain of Staphylococcus aureus in the presence of a beta-lactam antibiotic selected from cefoxitin and 6-APA (6-aminopenicillanic acid), at a temperature between 30° and 40° Celsius, for a period of between 15 and 40 minutes, b) isolating the bacteria present in said biological sample, c) lysing the bacteria and hydrolyzing the bacterial proteins, to obtain a mixture of peptides, d) analyzing this mixture of peptides by targeted mass spectrometry coupled with peptide separation, detecting at least one peptide derived from the PBP2a protein (SEQ ID NO. 1) or from the PBP2c protein (SEQ ID NO. 2).2) during the analysis step (d) being indicative of the methicillin resistance of the strain of Staphylococcus aureus present in said biological sample.

2. A method according to claim 1, characterized in that the separation of peptides is a separation by chromatography or by electrophoresis.

3. Method according to claim 1 or 2, characterized in that the mass spectrometry analysis is targeted during the analysis (SRM / MRM, MRM3, PRM type mode) or after the analysis (DIA / SWATH type mode).

4. A method according to any one of claims 1 to 3, characterized in that step (b) also includes a step of selective lysis of non-bacterial cells present in the biological sample.

5. A method according to any one of claims 1 to 4, characterized in that the hydrolysis of bacterial proteins in step (c) is an enzymatic hydrolysis.

6. A method according to claim 5, characterized in that the enzyme is trypsin.

7. A method according to any one of claims 1 to 6, characterized in that the at least one detected peptide is chosen from the group of 12 peptides having the following sequences: SEQ ID NO. 3 to SEQ ID NO.

14.

8. A method according to any one of claims 1 to 7, characterized in that

9. The biological sample is chosen from: - A biological fluid such as blood, serum, plasma, urine, cerebrospinal fluid, and tears; - A bacterial culture such as blood culture, colony on agar, culture broth; - A food sample, and - any other type of biological sample. Kit for implementing the process according to any one of claims 1 to 8, including: - at least one antibiotic from the beta-lactam class chosen from cefoxitin and 6-APA (6-aminopenicillanic acid), - trypsin, and - a reagent allowing selective lysis of non-bacterial cells present in the biological sample, for example a detergent.

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