Method for Determining Methicillin Resistance in Staphylococcus aureus Strains

Abstract

The present invention relates to a method for determining the methicillin resistance characteristic of Staphylococcus aureus strains present in a biological sample, comprising the following steps: a) incubating a biological sample containing a Staphylococcus aureus strain in the presence of an antibiotic from the class of beta-lactams selected from the following group: cefoxitin and 6-APA (6-aminopenicillanic acid) for at least 15 minutes; b) isolating the bacteria present in the biological sample; c) lysing the bacteria and hydrolyzing the bacterial proteins in order to obtain a mixture of peptides; and d) analysing this mixture of peptides by targeted mass spectrometry, wherein the detection of at least one peptide originating from protein PBP2a (SEQ ID NO: 1) or PBP2c (SEQ ID NO: 2) during this analysis step indicates methicillin resistance of the Staphylococcus aureus strain present in the biological sample.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to a method for characterizing bacterial strains belonging to the family Staphylococcus, in particular for identifying bacterial strains resistant to certain antibiotics, which allows medical personnel to select the appropriate antibiotic for each infected patient based on this resistance data. [Background technology]

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

[0003] The first penicillin-resistant strains of Staphylococcus aureus were characterized only 2 years after the introduction of this antibiotic into the range of treatments against bacterial infections (Kirby, 1944) and spread rapidly, leading to the gradual discontinuation of the use of penicillin in clinical practice in favor of new semisynthetic antibacterial molecules, such as methicillin and oxacillin.

[0004] Then, in the 1960s, new strains of Staphylococcus aureus that were resistant to methicillin as well as to other antibiotic compounds emerged. These strains are designated by the acronym MRSA, for Methicillin-Resistant Staphylococcus aureus.

[0005] These MRSA strains express a specific toxin, Panton-Valentine leukocidin (PVL), a so-called pore-forming toxin that induces pores in the cell walls of cells, particularly those of the immune system, thus killing cells that could fight infection.

[0006] Initially confined to hospital settings, MRSA strains have then spread throughout the population. In 2019, it was estimated that MRSA strains accounted for between 1% and 46% of Staphylococcus aureus infections in Europe. According to the World Health Organization, people infected with MRSA strains are 64% more likely to die than those infected with non-resistant strains.

[0007] Therefore, detection of MRSA strains is a major challenge for the management and treatment of patients with Staphylococcus aureus infection.

[0008] The MRSA phenotype is due to the expression of a specific protein, penicillin-binding protein 2a (PBP2a), encoded by the MecA gene, and, in rarer cases, the PBP2c protein, encoded by the MecC gene. These proteins replace the transpeptidase PBP2, an enzyme involved in bacterial cell wall formation. Since the alternative proteins PBP2a and PBP2c have 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 emergence of the first MRSA strains, several methods have been developed to detect this resistance trait, following three main approaches: - Phenotypic methods are based on the assessment 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 strain studied; - Immunological and proteomic methods are based on the detection of the PBP2a protein or peptides thereof. Among these techniques, immunochromatography (detection using antibodies) and mass spectrometry are particularly used.

[0010] These different approaches are briefly presented below.

[0011] phenotypic methods The traditional agar diffusion method is old but still widely used in laboratories due to its simplicity and low cost. It consists of culturing bacteria on agar in the presence of an antibiotic disk, usually oxacillin or cefoxitin. The susceptibility or resistance phenotype of the bacterial strain is determined by evaluating the diameter of the growth inhibition zone around the disk.

[0012] The main drawback of this technique is the time it takes to obtain results, approximately 12–18 h (Felten, 2002).

[0013] The arrival on the market of instruments such as the Vitek® 2 distributed by bioMerieux or the Phoenix™ distributed by Becton Dickinson allows high-throughput automation of antibiotic susceptibility testing. Although more robust than the agar diffusion method, these tests are also limited by the growth time of the bacteria. In the case of Staphylococcus aureus, results are obtained in about 10 to 13 hours.

[0014] For example, an article by Sparbier et al., 2013, discloses such a phenotypic method in which bacterial growth is evaluated in the presence of antibiotics. Bacteria are incubated in the presence of oxacillin or cefoxitin and a high molecular weight "heavy" lysine. Incorporation of this heavy amino acid is observed only in newly synthesized proteins. However, only bacteria resistant to the antibiotic show significant protein biosynthetic activity. Proteins containing heavy lysine are then detected by MALDI-TOF.

[0015] The main drawback of this so-called phenotypic technique is its sensitivity to factors that may affect bacterial growth, such as inoculum volume, incubation time and temperature, medium, pH or salt concentration (Tenover et al., 1999).

[0016] molecular biology methods The advent of molecular biology methods has significantly improved the identification time of MRSA strains compared to phenotypic methods. The high sensitivity and specificity of tests currently on the market make the detection of the MecA gene by polymerase chain reaction (PCR) the gold standard for the diagnosis of MRSA. Commonly used tests include the GeneXpert MRSA / SA BC distributed by Cepheid, the GeneOhm™ StaphSR Assay distributed by Becton Dickinson, the eazyplex® MRSAplus distributed by AmplexDiagnostics, or the FilmArray BCID distributed by bioMerieux. These tests allow the detection of MRSA strains in 1-3 hours directly from positive blood culture bottles, without the need for a prior subculture step consisting of isolating the bacteria and growing them.

[0017] The main drawback of this technique is that it is prone to generating false positives, especially for strains harboring an SSCmec cassette that does not contain the MecA gene, or for strains expressing variant proteins encoded by genes with sequences that differ slightly from the one established for the MecA gene.

[0018] Moreover, the high cost of molecular biology assays remains a major obstacle to their routine use.

[0019] Immunoassay: Detection of PBP2A protein The use of latex beads coupled with a specific monoclonal antibody that detects the PBP2a protein makes it possible to see with the naked eye agglutination that indicates the presence of the antigen in the sample. This is the principle on which the Oxoid PBP2' test distributed by Thermo Fisher Scientific or the Mastalex-MRSA test distributed by Mast Diagnostic are based.

[0020] The duration of the test itself consists of between 15 and 20 minutes. The test works very well, with a sensitivity of 100% and a specificity of greater than 99%.

[0021] The main drawback of this technique is that the 1.5 × 10 9 It requires a minimal amount of bacteria and involves a bacterial culture step lasting at least 10 hours.

[0022] Furthermore, these tests do not allow the detection of MRSA strains expressing the PBP2c protein that is not recognized by certain monoclonal antibodies used, which has been shown in particular for the Oxoid PBP2' test (Dupieux et al., 2017).

[0023] Immunochromatographic tests for the detection of MRSA strains are also commercially available, such as the Clearview™ PBP2a SA Culture Colony Test kit distributed by the company Alere.

[0024] Although very rapid and with a sensitivity and specificity greater than 98%, this test also requires a bacterial culture step.

[0025] The Clearview™ PBP2a SA Culture Colony Test is advantageously able to detect PBP2c from colonies growing on the edge of a cefoxitin disk after agar incubation, however, the induction step required for detection of MecC results in an additional delay of approximately 18 hours before results are provided (Dupieux et al., 2017).

[0026] Targeted Mass Analysis Recent studies have highlighted the potential of so-called bottom-up, ie proteomic approaches based on targeted mass spectrometric peptide fragmentation spectra, for pathogen identification and demonstration of resistance mechanisms.

[0027] These approaches include the following steps: 1) Mechanical or chemical lysis of bacterial samples to release intracellular proteins; 2) enzymatic digestion of bacterial proteins using a protease (typically trypsin) to generate a mixture of peptides; 3) chromatographic separation coupled with targeted mass spectrometry of the samples, where the mass spectrometer operates in MS / MS mode for the detection of marker peptides of the resistant proteins; 4) Reprocessing of the results to verify the presence / absence of the marker peptide in the sample.

[0028] The term "targeted" indicates that specific peptide sequences are sought; these can be the entire protein sequence, or peptides derived from enzymatic digestion of the protein.

[0029] Targeted mass spectrometry has been successfully used to characterize the resistance and virulence of Staphylococcus aureus strains, in particular by detecting the PBP2a protein or virulence factors, such as PVL (Charretier et al., 2015).

[0030] International application WO 2011 / 045544 describes a method involving the characterization of resistance and virulence of Staphylococcus aureus by liquid chromatography coupled with targeted mass spectrometry, in which the starting biological material is isolated colonies on an agar medium.

[0031] 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 technique based on the analysis of the fragmentation spectrum of the whole protein). In the absence of an enzymatic protein digestion step, the time required to obtain a result is reduced (Neil et al., 2021). However, the authors did not demonstrate the applicability of their method to the detection of PBP2a directly in blood culture samples without a prior subculture step. [Prior art documents] [Patent documents]

[0032] [Patent Document 1] WO 2011 / 045544 Summary of the Invention [Problem to be solved by the invention]

[0033] To date, no technology is known that meets all the criteria required for installation in a hospital, in particular short analysis time, simplicity of sample preparation, limited cost and satisfactory performance.

[0034] To meet the current demands of analytical laboratories, methods for identifying MRSA strains must meet at least the following criteria: - no subculture step of bacteria isolated from blood (in case of positive detection, analysis directly from blood culture bottle); - short sample preparation and analysis times (less than 2 hours) in order to obtain results quickly and thus enable the administration of appropriate antibiotic treatment to the patient in the shortest possible time; - Low cost so that the tests can be routinely carried out; - a test that detects both proteins PBP2a and PBP2c, including variant proteins with peptide sequences that have sequence identity to SEQ ID NO: 1 or 2, with a sensitivity and specificity approaching 100%.

[0035] We have developed a targeted method for the detection of MRSA strains by liquid chromatography coupled to mass spectrometry, performed directly from positive blood culture bottles, including an induction step and rapid sample preparation, allowing results to be obtained in less than 1.5 hours from the detection of the presence of Staphylococcus aureus strains in a biological sample. [Means for solving the problem]

[0036] Disclosure of the Invention The present invention relates to a method for determining the methicillin resistance profile of a strain of Staphylococcus aureus present in a biological sample, comprising the following steps: a) incubating the biological sample containing the S. aureus strain in the presence of an antibiotic from the beta-lactam class selected from the following group: cefoxitin and 6-aminopenicillanic acid (6-APA) for at least 15 minutes; b) isolating the bacteria present in the biological sample; c) lysing the bacteria and hydrolysing the bacterial proteins to obtain a mixture of peptides; d) analyzing this mixture of peptides by targeted mass spectrometry Including, The method further relates to a method, wherein the detection of at least one peptide derived from the PBP2a (SEQ ID NO: 1) or PBP2c (SEQ ID NO: 2) protein during said analysis step is indicative of methicillin resistance of a Staphylococcus aureus strain present in said biological sample.

[0037] The present invention relates to - antibiotics of the beta-lactam class selected from the following group: cefoxitin and 6-aminopenicillanic acid (6-APA); - Trypsin; - a reagent allowing the 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 targeted mass spectrometry The present invention also relates to a kit for carrying out the method, comprising: Description of the drawings [Brief description of the drawings]

[0038] [Figure 1]Figure 1 shows chromatograms obtained from MRM analysis of MRSA26b strain. A) Sample prepared without induction step. B) Sample prepared with induction step. Peaks corresponding to transitions of the PBP2a peptide are indicated by arrows. [Diagram 2] Figure 2 shows chromatograms obtained from the MRM3 analysis of MRSA26b strain. A1 and A2) MRM3 chromatograms of VALELGSK (A1) and FQITTSPGSTQKK (A2) peptides for samples prepared without a derivatization step. B1 and B2) MRM3 chromatograms of VALELGSK (B1) and FQITTSPGSTQKK (B2) peptides for samples prepared with a derivatization step. [Diagram 3] Figure 3 shows chromatograms obtained from MRM analysis of MRSA28b strain. A) Sample prepared without induction step. B) Sample prepared with induction step. Peaks corresponding to transitions of the PBP2a peptide are indicated by arrows. [Figure 4] Figure 4 shows chromatograms obtained from MRM3 analysis of MRSA28b strain. A1 and A2) MRM3 chromatograms of VALELGSK (A1) and FQITTSPGSTQKK (A2) peptides for samples prepared without induction step. B1 and B2) MRM3 chromatograms of VALELGSK (B1) and FQITTSPGSTQKK (B2) peptides for samples prepared with induction step. [Diagram 5] Figure 5 shows the chromatograms of the MSSA16b strain after the induction step. A) MRM chromatogram. B1 and B2) MRM3 chromatograms for the VALELGSK (B1) and FQITTSPGSTQKK (B2) peptides. For both types of analysis, no peptide is detected. [Figure 6] Figure 6 shows chromatograms of the MSSA1b strain after the induction step. A) MRM chromatogram. B1 and B2) MRM3 chromatograms for the VALELGSK (B1) and FQITTSPGSTQkk (B2) peptides. For both types of analysis, no peptides are detected. [Figure 7]Figure 7 shows chromatograms obtained from MRM analysis of strains expressing PBP2c. A) Sample prepared without induction step. B) Sample prepared with induction step. Peaks corresponding to transitions of the PBP2c peptide are indicated by arrows. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] Detailed Description of the Invention The present invention relates to a method for detecting methicillin-resistant strains of Staphylococcus aureus, referred to as MRSA strains, that allows results to be obtained in less than 1.5 hours from a biological sample identified as containing a strain of S. aureus.

[0040] Methicillin, also spelled methicillin, is a beta-lactam antibiotic that belongs to the penicillin subfamily. Its CAS number is 61-32-5. It was widely used against Staphylococcus aureus infections before being replaced by cloxacillin, which is less likely to develop bacterial resistance.

[0041] Advantageously, the method of the invention makes it possible to identify strains resistant to this antibiotic (MRSA) with a sensitivity and specificity approaching 100%.

[0042] The method according to the invention uses biological samples without a bacterial subculture step, such as positive blood culture samples (containing blood cells and bacteria). The first step of induction of expression of PBP2a or PBP2c protein using an antibiotic of the beta-lactam class is followed by a step of rapid isolation of the bacteria, then a step of bacterial lysis and enzymatic digestion of the proteins, and finally a targeted mass spectrometry analysis for detection of peptides derived from the enzymatic digestion of the PBP2a or PBP2c protein.

[0043] The present invention therefore relates to a method for determining the methicillin resistance profile of a strain of Staphylococcus aureus present in a biological sample, comprising the following steps: a) incubating the biological sample containing the Staphylococcus aureus strain in the presence of an antibiotic from the beta-lactam class selected from the following group: cefoxitin and 6-aminopenicillanic acid (6-APA) for at least 15 minutes; b) isolating the bacteria present in the biological sample; c) lysing the bacteria and hydrolysing the bacterial proteins to obtain a mixture of peptides; d) analyzing this mixture of peptides by targeted mass spectrometry Includes; The method further comprises the step of: detecting at least one peptide derived from the PBP2a or PBP2c protein in said assay step, which is indicative of resistance to methicillin of a strain of Staphylococcus aureus present in said biological sample.

[0044] This method was developed from the method described in international application WO 2011 / 045544.

[0045] Based on this method, we identified that the PBP2a protein is highly heterogeneously expressed among MRSA strains, with widely differing levels of expression.

[0046] Using a methodology similar to that presented in application WO 2011 / 045544, but using blood culture media positive for Staphylococcus aureus as starting biological sample, i.e. without a bacterial subculture step, it has been demonstrated for a cohort of 98 MRSA strains representative of French epidemiology that approximately 60% of the MRSA strains have PBP2a expression levels that are too low to be detected by the described methodology (see experimental part).

[0047] Indeed, heterogeneity of PBP2a expression exists among different MRSA strains: some strains that naturally express PBP2a at high levels (detectable without induction) have very low basal expression levels that do not allow detection of PBP2a by direct analysis without an induction step.

[0048] It was therefore concluded that the technique described in application WO 2011 / 045544 is not suitable for implementation in clinical analytical laboratories, since many MRSA strains cannot be detected.

[0049] The present application relates to an improvement to the method, which includes the addition of step (a), incubating the biological sample containing the S. aureus strain in the presence of a beta-lactam antibiotic for at least 15 minutes to induce expression of the PBP2a or PBP2c protein, thus having sufficient new expression to detect the variant protein expressed in 100% of MRSA strains.

[0050] The PBP2a protein has the following sequence: Sequence 1, PBP2a, Staphylococcus aureus (NCBI ID: WP_001801873.1): [ka]

[0051] The PBP2c protein has the following sequence: Sequence 2, PBP2c, Staphylococcus aureus (NCBI ID: WP_000725529.1): [ka]

[0052] Advantageously, the method of the invention makes it possible to detect the two PBP2a and PBP2c proteins as well as variant proteins.

[0053] The term "variant protein" is understood to mean a protein having a peptide sequence that has strong sequence identity with the sequence SEQ ID NO: 1 or 2, in particular at least 90%, or better still at least 95%, or even 99% sequence identity with one of the sequences SEQ ID NO: 1 or SEQ ID NO: 2. Variant proteins generally have one, two or three point mutations in the wild-type protein sequence, i.e. they differ by only one, two or three amino acids in the peptide sequence.

[0054] The percentage of identity to which reference is made in the context of the present disclosure is determined after optimal alignment of the sequences has been compared and may therefore include one or more additions, deletions, truncations and / or substitutions.

[0055] This percentage of identity can be calculated by any sequence analysis method known to those skilled in the art.

[0056] The percentage of identity can be determined after global alignment of the sequences to be compared, carried out in their entirety, over their entire length. In addition to manual methods, it is possible to determine the global alignment of sequences by the algorithm of Needleman and Wunsch (1970).

[0057] For amino acid sequences, sequences can be compared using any software known to those skilled in the art, such as, for example, the Needle software. The parameters used can include, in particular: "Gap open" equal to 10.0, "Gap extension" equal to 0.5, and the BLOSUM62 matrix.

[0058] Preferably, the percentage of identity defined in the context of the present invention is determined by global alignment of the sequences compared over their entire length.

[0059] (a) Inducing expression of PBP2a or PBP2c Induction consists of carrying out a rapid incubation (in particular lasting less than 3 hours, preferably less than 1 hour) in the presence of the antibiotic to activate the system regulating the expression of the PBP2a or PBP2c protein, thus resulting in overexpression of these proteins.

[0060] This incubation of the biological sample containing the strain of Staphylococcus aureus in the presence of an antibiotic of the beta-lactam class selected from cefoxitin and 6-aminopenicillanic acid (6-APA) for at least 15 minutes induces the expression of at least one protein selected from PBP2a and PBP2b in the bacterial strain.

[0061] The antibiotic used is of the beta-lactam class, i.e. an antibiotic containing a beta-lactam ring, and is selected from cefoxitin and 6-aminopenicillanic acid (6-APA).

[0062] Cefoxitin, CAS number 35607-66-0, is an antibiotic of the beta-lactam family, classified as a class of so-called second-generation cephalosporins. Cefoxitin exerts its bactericidal action by inhibiting cell wall synthesis.

[0063] 6-Aminopenicillanic acid, abbreviated as 6-APA, CAS number 551-16-6, is a derivative of penicillin.

[0064] Preferably, the antibiotic used in the induction step (a) is cefoxitin.

[0065] Induction of PBP2a or PBP2c expression is typically performed by incubation of the biological sample in the presence of an appropriate amount of antibiotic at a temperature comprised between 30 and 40 degrees C. Incubation of the biological sample is preferably performed at 37° C. with agitation.

[0066] According to a particular implementation of the method of the invention, the incubation step (a) is carried out for a period of at least 15 minutes.

[0067] Preferably, this incubation step (a) is carried out for a period of less than 3 hours, or less than 2 hours, or preferentially less than 1 hour.

[0068] Advantageously, the incubation time may be at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes or at least 45 minutes.

[0069] In particular, this incubation step (a) is carried out for one of the following durations: 15-180 minutes; 15-120 minutes; 15 to 90 minutes; or 15 to 60 minutes.

[0070] As shown in the examples, in the absence of this step of inducing expression of PBP2a or PBP2c, some methicillin-resistant S. aureus strains would not be identified as such because detection of one of the PBP2a or PBP2c proteins is not possible due to too low expression of the protein in the strain.

[0071] Step (b) of isolating the bacteria The bacteria may be isolated by any means known to those skilled in the art, in particular by centrifugation.

[0072] According to one implementation of the method, step (b) also comprises a step of selective lysis of non-bacterial cells present in the biological sample, this selective lysis step being carried out prior to centrifugation of the sample.

[0073] Detergent compounds allow the lysis of animal cells by dissociation of the membrane. Bacteria are composed of a rigid peptidoglycan wall and are therefore not lysed by the action of detergents.

[0074] The detergent compound may for example be selected from the group of saponin, Triton X100 or sodium dodecyl sulfate (SDS).

[0075] In particular, when the biological sample is a blood sample, step (b) of isolating the bacteria is advantageously carried out simultaneously with the lysis of blood cells present in the sample by the addition of a surfactant compound.

[0076] (c) lysing the bacteria and hydrolysing bacterial proteins; Various methods for lysing bacterial cells can be used: examples include thermal methods (temperatures 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).

[0077] According to a preferred implementation of the method of the invention, the bacteria are lysed by sonication.

[0078] Hydrolysis of bacterial proteins to peptides is usually carried out by one of two processes: a) by the action of chemical agents, e.g., hydroxyl groups, which induce random cleavage at the level of peptide bonds; or b) By the action of proteolytic enzymes (proteases) which act by hydrolysis of protein bonds.

[0079] According to a preferred implementation of the method, the hydrolysis of bacterial proteins in step (c) is an enzymatic hydrolysis.

[0080] Commonly used proteases include pepsin, which hydrolyzes peptide bonds preferentially before aromatic amino acids (tyrosine, tryptophan and phenylalanine), GluC endoprotease, which cleaves peptide bonds at glutamic acid residues, or trypsin, which cleaves proteins on the C-terminal side of the amino acids lysine and arginine.

[0081] According to a preferred implementation of the method of the present invention, trypsin is used as the enzyme for the bacterial protein hydrolysis step. Trypsin is preferred due to the specific nature of its activity, the appropriate size of the peptides it produces, and the tendency of tryptic peptides to have a positively charged amino acid (lysine or arginine) at the C-terminus, thus facilitating analysis by mass spectrometry (analysis of charged molecules).

[0082] A specific protocol for step (c) is given in the experimental part.

[0083] Step (d) of analysis by targeted mass spectrometry Mass spectrometry is a physical analytical technique for detecting and identifying molecules of interest. It is also known as single reaction monitoring, multiple reaction monitoring, or parallel reaction monitoring. Its principle is the gas-phase separation of charged molecules (ions) according to their mass / charge ratio (m / z).

[0084] Mass spectrometers are i) an ionization source for ionizing the molecules to be analyzed, i.e., giving them a positive or negative charge; ii) a mass analyzer for separating the ionized molecules according to their mass to charge ratio (m / z); iii) a detector for measuring the signal generated either directly by the molecular ions or by ions produced from the molecular ions, as described below. Includes.

[0085] The ionization step necessary to perform mass spectrometry can be performed by any method known to those skilled in the art. The ionization source makes it possible to bring the molecules to be assayed into a gaseous, ionized state. The ionization source can be used in a positive mode to study positive ions, or in a negative mode to study negative ions. Several types of sources exist and are used depending on the desired results and the molecules to be analyzed.

[0086] The mass analyzer in which the step of separating the ionized markers as a function of their mass / charge ratio (m / z) is carried out can be any mass analyzer known to those skilled in the art. Examples include low-resolution analyzers, such as quadrupole (Q), 3D ion trap (IT) or linear ion trap (LIT), and high-resolution analyzers that measure the exact mass of the analytes and use a magnetic sector coupled to an electric sector, time of flight (TOF), or Orbitrap.

[0087] The separation of the molecular ions as a function of their m / z ratio can be performed once (simple mass spectrometry or MS) or several successive MS separations can be performed. If two successive MS separations are performed, the analysis is called MS / MS or MS2. If three successive MS separations are performed, the analysis is called MS / MS / MS or MS3.

[0088] Targeted mass spectrometry is a variation in which the molecules of interest sought by the analytical technique are known in advance and the analysis is used to identify whether they are present in a sample.

[0089] The targeted approach (multiple reaction monitoring, MRM; parallel reaction monitoring, PRM; multiple reaction monitoring high resolution, MRM-HR; multiple reaction monitoring cubed, MRM3, DIA / SWATH) consists of the selection by a first analyzer of the exact 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.

[0090] In the case of chromatographic coupling, the signals of the fragments are measured as a function of time which are presented in the form of a chromatogram; thus, the appearance of simultaneous chromatographic peaks (simultaneous detection of fragments) indicates the presence of the peptide in the sample.

[0091] These approaches require preliminary selection and validation of marker peptides to ensure their sequence / mass uniqueness (or mass / charge ratio, m / z) and their detectability.

[0092] The principle of SRM mode or of MRM mode is to specifically select a precursor ion, fragment it and then specifically select one of the fragment ions. For such applications, triple quadrupole or triple quadrupole hybrid with ion trap devices are commonly used.

[0093] The DIA / SWATH analysis mode consists of recording fragmentation spectra of successive precursor ion selection windows, usually overlapping by one unit of mass to charge ratio (m / z), 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 covering all these windows allows each chromatographic peak to be sampled for at least five points.

[0094] According to a preferred embodiment of the invention, mass spectrometry is targeted during the analysis (SRM / MRM, MRM, MRM3 or PRM mode) or after the analysis (DIA / SWATH mode).

[0095] More preferably, the targeted mass spectrometric analysis is carried out in MRM or MRM3 mode, most preferably in MRM3 mode.

[0096] Targeted mass spectrometry is preferably coupled to separation of the peptides by chromatographic or electrophoretic separation of the peptides.

[0097] Separation of the peptides can be carried out by any technique known to those skilled in the art, in particular by reversed-phase liquid chromatography, by normal-phase liquid chromatography, by hydrophilic-phase liquid chromatography or by capillary electrophoresis.

[0098] Preferably, separation of the peptides is carried out by reverse phase liquid chromatography.

[0099] The peptides sought during this analysis step are peptides derived from the PBP2a or PBP2c proteins that are specific for these proteins.

[0100] It will be understood that at least one peptide derived from these proteins may be detected, but preferably several peptides are detected during the analysis to confirm the results obtained.

[0101] According to a preferred implementation of the method of the present invention, at least one peptide derived from the PBP2a or PBP2c protein to be detected, i.e. the presence of which is detected by targeted mass spectrometry, is selected from the group consisting of 12 peptides having the following sequences as shown in Tables 1 and 2 below: SEQ ID NO: 3 to SEQ ID NO: 14.

[0102] [Table 1]

[0103] [Table 2]

[0104] Advantageously, all these peptides were selected in conserved regions of the proteins and are therefore also identified in variant proteins of PBP2a and PBP2c.

[0105] The method according to the invention can be carried out on any type of biological sample capable of containing a Staphylococcus aureus strain.

[0106] According to a particular implementation of the method, the biological sample comprises: - Biological fluids, such as blood, serum, plasma, urine, cerebrospinal fluid and tears; - Bacterial cultures, e.g. blood cultures, bacterial colonies on agar, bacterial culture broth; - Food samples; and - any other type of biological sample is selected from.

[0107] This is preferably a biological fluid, in particular a blood sample (blood, serum, plasma), in particular a medium of a positive blood culture containing Staphylococcus aureus.

[0108] The term "blood culture" is understood to mean a blood sample taken from a patient and then incubated in appropriate conditions allowing the growth of any bacteria present in the sample.

[0109] The blood cultures may be performed in blood culture bottles, such as those commercially available in the Bact / Alert range distributed by bioMerieux, or in the Bactec range distributed by Becton Dickinson.

[0110] Kits for carrying out the methods The present invention relates to - antibiotics of the beta-lactam class selected from the following group: cefoxitin and 6-aminopenicillanic acid (6-APA); - Trypsin; - Reagents that allow the selective lysis of non-bacterial cells present in the biological sample, e.g. detergents The present invention also relates to a kit for carrying out the above method, comprising:

[0111] For example, reagents for enabling the selective lysis of non-bacterial cells include detergent compounds selected from the following group of compounds: saponin, Triton X100 or sodium dodecyl sulfate (SDS). EXAMPLES

[0112] Working Example The examples presented below are intended to illustrate the methods according to the present invention but in no way limit the scope of the present invention.

[0113] In particular, in the examples shown below, the antibiotic used during the induction step is cefoxitin, although it will be appreciated that 6-APA may also be used.

[0114] Example 1 Materials and Methods The induction step (a) is carried out according to the following protocol: - Disinfect the septum of the blood culture bottle. - Using a 21 G syringe and needle, remove 3.8 mL of positive blood culture media and transfer to a 15 mL tube. - Add 200 μL of 8 μg / mL cefoxitin solution. - Homogenize the mixture and incubate the tubes for 30 minutes at 37°C with agitation (180 rpm).

[0115] Step (b) of isolating the bacteria is advantageously carried out simultaneously with the lysis of the blood cells, according to the following protocol: - After 30 minutes of induction, transfer 1 mL of medium into a 1.5 mL tube, add 200 μL of 12% SDS solution and vortex for 10 seconds. - Centrifuge at 16,100 g for 2 minutes and remove the supernatant. - Resuspend the pellet in 1 mL of saline. - Centrifuge at 16,100g for 1 minute and discard the supernatant. - Resuspend in 1 mL of saline.

[0116] Step (c) of mechanical lysis of the bacteria and enzymatic digestion of the proteins is carried out according to the following protocol: - Transfer 200 μL 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 1 mg / mL trypsin solution extemporaneously prepared from lyophilized trypsin in 150 mM ammonium bicarbonate buffer. The sample is immediately placed in a sonicator water bath set at -50°C and then 10 sonication cycles (low power) are initiated. 〇 Ultrasound on for 30 seconds 〇 Ultrasonic off for 30 seconds - Add 5 μL of formic acid directly at the end of the 10 sonication cycles. - Centrifuge the tubes at 9600g for 5 minutes. - Transfer 150 μL of the supernatant into a 2 mL amber glass vial with an insert for mass spectrometry analysis.

[0117] Step (d) of the targeted mass analysis is carried out according to the following protocol: A volume of 5 μL of sample from the previous dissolution / digestion step is injected into the chromatographic system. Analysis is performed on a Waters XBridge Peptide BEH C18 reversed-phase column, 1 mm inner 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 Autosampler, and an Agilent 1290 TCC column oven set at 60° C. The gradient used for the chromatographic separation is shown in Table 3.

[0118] Solvent A: HO + 0.1% formic acid Solvent B: Acetonitrile + 0.1% formic acid

[0119] [Table 3]

[0120] The outlet of the chromatography system is directly connected to the ionization source of a QTRAP 6500+ mass spectrometer (Sciex) for online analysis of peptides from bacterial protein digests. The mass spectrometer was operated in MRM or MRM-cubed (MRM3) mode and the transitions monitored for PBP2a protein are listed in Table 4.

[0121] [Table 4A] [Table 4B]

[0122] The transitions followed for the PBP2c protein are listed in Table 5.

[0123] [Table 5]

[0124] The mass spectrometer parameters for analysis in MRM mode are listed in Table 6 below.

[0125] [Table 6]

[0126] Mass spectrometer parameters for analysis in MRM3 mode are given below in Table 7 (peptide VALELGSK) and Table 8 (peptide FQITTSPGSTQK).

[0127] [Table 7]

[0128] [Table 8]

[0129] Example 2 First result 1 and 2 show the necessity of a derivatization step for detection of peptides derived from PBP2a by MRM.

[0130] Chromatogram A results from the analysis of the strain without an induction step, chromatogram B corresponds to the analysis of the same strain after an induction step.

[0131] In the absence of induction, the peptide peaks derived from PBP2a are confused with background noise and are therefore undetectable.

[0132] In contrast, after the induction step, characteristic peaks are observed: they are precisely delineated and rise above the background noise. PBP2a is precisely detected.

[0133] Blood cultures were inoculated with 98 strains of MRSA and 19 strains of Methicillin-susceptible Staphylococcus aureus (MSSA) that do not express PBP2a, and samples were then prepared and analyzed in bottle positives according to the protocol in Example 1.

[0134] The area under the curve was measured for each transition. The area under the curve was also measured for MSSA-susceptible strain samples that do not express PBP2a but over the peptide elution window to obtain a baseline value. Because the signal of the transition can be contaminated by noise or interference due to the matrix, it is necessary to evaluate this baseline in a matrix that does not contain the analyte (here PBP2a) in order to establish a threshold above which we can confirm the presence of the peptide.

[0135] For each transition, the threshold corresponds to 150% of the maximum area measured over the peptide elution window for the MSSA control sample.

[0136] Example 3 MRM spectroscopy results All MRM transitions with area values ​​greater than the threshold were considered positive and are indicated in the results table below as 1. Transitions with areas less than the threshold were indicated as 0 and are therefore considered negative. A sample was considered to contain an MRSA strain if at least three peptides were detected in at least two positive transitions.

[0137] The following Tables 9 and 10 correspond to the results obtained during the MRM analysis of 98 MRSA without the derivatization step.

[0138] Tables 11 and 12 below correspond to the results obtained during the MRM analysis of 98 MRSA with the derivatization step.

[0139] [Table 9A] [Table 9B]

[0140] [Table 10A] [Table 10B]

[0141] [Table 11A] [Table 11B]

[0142] [Table 12A] [Table 12B]

[0143] Based on the results shown in Tables 9 to 12, the following conclusions can be stated: Without the induction step, 38 out of 98 MRSA showed at least two positive transitions for at least three peptides, which corresponds to a detection sensitivity of 39%.

[0144] With the induction step, 96 out of 98 MRSA have at least two positive transitions for at least three peptides, which corresponds to a sensitivity of 98% detection of MRSA by these validation criteria. Only MRSA9b and MRSA41b strains are not correctly identified, because for MRSA41b strain only two peptides are detected in two transitions and for MRSA9b strain one peptide is detected in two transitions.

[0145] All MSSA analyzed using the same method have area under the curve values ​​for each transition lower than the previously set thresholds, meaning that the MSSA are not identified as MRSA, which is equivalent to 100% specificity.

[0146] Example 4 MRM3 spectroscopic analysis results for PBP2a MRM3 transitions with area values ​​greater than the threshold are considered positive and are designated as 1. Transitions with areas less than or equal to the threshold are designated as 0 and considered negative. A sample was considered identified as MRSA if at least one MRM3 transition was detected as positive. 19 MSSA strains were analyzed following the same procedure and the results are shown in the table below.

[0147] Table 13 corresponds to the results obtained during the MRM3 analysis of 98 MRSA without an induction step, which shows the validation of the MRM3 transitions of 98 MRSA prepared without an induction step.

[0148] Table 14 corresponds to the results obtained during the MRM3 analysis of 98 MRSA with induction step. It shows the validation of the MRM3 transitions of 98 MRSA prepared with induction step.

[0149] [Table 13]

[0150] [Table 14]

[0151] Nineteen methicillin-susceptible strains (MSSA1b, 2b, 3b, 4b, 5b, 6b, 7b, 8b, 9b, 10b, 11b, 12b, 13b, 14b, 15b, 16b, 17b, 18b and 19b) were tested: as expected, no peptides derived from PBP2a were detected. Results with all zero values ​​are not shown in detail.

[0152] Without the induction step, 87 out of 98 strains showed at least one positive transition, which corresponds to a sensitivity of 89% (Table 12).

[0153] With the induction step, all MRSA strains tested had at least one positive MRM3 transition, which corresponds to a sensitivity of 100% (Table 13).

[0154] All MSSA analyzed following the same method (with induction step) have area under the curve values ​​for each transition that are less than or equal to the pre-determined threshold, meaning that no susceptible MSSA strains are identified as MRSA, which equates to 100% specificity.

[0155] conclusion The performance of the method according to the invention is shown in Table 15 below, which illustrates the importance of the induction step.

[0156] [Table 15]

[0157] These results show that the method according to the invention allows the rapid identification of MRSA directly from a positive blood sample (blood culture) in less than 1.5 hours, with a performance superior to other methods currently on the market (98% sensitivity and 100% specificity for MRM, 100% sensitivity and 100% specificity for MRM3).

[0158] The sample preparation protocol is simple to implement and the cost of consumables per analysis is minimal.

[0159] Furthermore, since the analysis is based on the detection of eight different peptides of the PBP2a protein, the possibility of non-detection in the case of PBP2a variant proteins carrying one or more point mutations is very low.

[0160] Finally, it has also been shown that the induction step of the method is necessary for the detection of the PBP2c protein in strains of Staphylococcus aureus expressing that protein. The results shown in Figure 7 show that detection was not possible without a prior step of induction by incubation with an antibiotic of the beta-lactam family.

[0161] References [Table 16]

Claims

1. 1. A method for determining the methicillin resistance characteristics of strains of Staphylococcus aureus present in a biological sample, comprising the steps of: a) incubating the biological sample containing the Staphylococcus aureus strain in the presence of an antibiotic from the beta-lactam class selected from cefoxitin and 6-aminopenicillanic acid (6-APA) for at least 15 minutes; b) isolating bacteria present in said biological sample; c) lysing the bacteria and hydrolyzing bacterial proteins to obtain a mixture of peptides; d) analyzing this mixture of peptides by targeted mass spectrometry coupled with peptide separation Includes; The method, wherein detection of at least one peptide derived from the PBP2a protein (SEQ ID NO: 1) or the PBP2c protein (SEQ ID NO: 2) during said analyzing step (d) indicates said methicillin resistance of said Staphylococcus aureus strain present in said biological sample.

2. 2. The method of claim 1, wherein the separation of the peptides is a chromatographic or electrophoretic separation.

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

4. 3. The method according to claim 1 or 2, characterized in that the incubation step (a) is carried out for a period of between 15 and 180 minutes.

5. 3. The method according to claim 1 or 2, characterized in that step (b) also comprises a step of selective lysis of non-bacterial cells present in the biological sample.

6. 3. The method according to claim 1 or 2, characterized in that the hydrolysis of the bacterial proteins in step (c) is an enzymatic hydrolysis.

7. 7. The method of claim 6, wherein the enzyme is trypsin.

8. 3. The method according to claim 1 or 2, characterized in that the at least one peptide to be detected is selected from the group of 12 peptides having the following sequences: SEQ ID NO: 3 to SEQ ID NO:

14.

9. the biological sample - biological fluids, such as blood, serum, plasma, urine, cerebrospinal fluid and tears; - Bacterial cultures, for example, blood cultures, bacterial colonies on agar, bacterial culture broth; - Food samples; and - any other type of biological sample 3. The method according to claim 1 or 2, characterized in that the compound is selected from the group consisting of:

10. - at least one antibiotic of the beta-lactam class selected from cefoxitin and 6-aminopenicillanic acid (6-APA); - trypsin; and - a reagent, such as a detergent, that allows the selective lysis of non-bacterial cells present in said biological sample 3. A kit for carrying out the method according to claim 1 or 2, comprising:

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