1
Method for characterizing at least one microorganism by means of mass spectrometry
The present invention relates to the field of microbiology. More specifically, the invention relates to the characterization of microorganisms from a sample using mass spectrometry.
Since the discovery of microbes by Pasteur, microorganisms have been studied by microscopy and biochemical analyses. These conventional methods are often long and laborious and analytical alternatives were very soon sought. Thus, the analysis of bacteria by mass spectrometry was initiated as early as 1975 by J. Anhalt andC. Fenselau [1].
These preliminary studies were followed by the study, by gas chromatography coupled to mass spectrometry (GC-MS), of microorganism wall fatty acids [2]. This method was popularized under the name FAME for Fatty Acid Methyl Ester. It currently constitutes a reference method for taxonomic studies. However, its use remains limited to certain specialist laboratories which master the treatment of the sample by saponification, hydrolysis and derivation.
In 1996, the work of M. Claydon et al. [3] and also of T. Krishnamurthy and P. Ross [4] showed the possibility of identifying various bacterial species with a mass spectrometer of MALDI-TOF (Matrix Assisted Laser Desorption Ionization - Time Of Flight) type. The analysis combines the acquisition of a mass spectrum and the interpretation by expert software. It is extremely simple and can be carried out in a few minutes. However, it has only very recently become to spread among medical test laboratories [5]. Its clinical use is currently limited to the identification of species of bacteria and yeasts. It is used neither for typing nor for identifying resistances to antimicrobials, nor for analyzing virulence.
However, the characterization of microorganisms is fundamental both in the clinical field and in the industrial field. Thus, for example, the identification of resistances to antimicrobials such as antibiotics, and the detection of virulence factors are essential elements for ensuring optimum treatment of patients. Likewise, typing is crucial for epidemiological studies and for combating nosocomial diseases.
Other methods of mass spectrometry, in particular tandem mass spectrometry, have been proposed in order to meet these needs. By way of example, mention may be made of the work of C. Fenselau et al. for identifying P-lactamase with a quadrupole-TOF (Q-TOF) [6], the work of D. Ding et al. for the detection of staphylococcal

2 enterotoxin C2 (virulence factor SEC2) with a triple quadrupole [7], or else the work of R. Everley et al. for the typing of Clostridium with a Q-TOF [8].
However, these research results are not applicable to routine clinical use. They were obtained with research instruments requiring highly qualified personnel. The analysis times, which are often more than one hour per sample, are incompatible with the workload of a microbiological test laboratory. Finally, the data obtained by the various teams reply to a specific question, but not simultaneously to all the clinical needs.
More recently, S. Hofstadler et al. have proposed a method which meets all the clinical needs [9]. They have combined amplification of the microbial genome by PCR with detection of the PCR products by electrospray-TOF (ESI-TOF). This method is now completely automated [10]. However, it requires a PCR amplification with the deficiencies inherent in molecular biology, namely cost of probes, extraction yield, etc.
In this context, the objective of the present invention is to propose a method for characterizing microorganisms, namely identifying and determining the properties of typing, resistance to at least one antimicrobial, and virulence factor, which makes it possible to overcome the drawbacks of the prior art methods, namely to provide a method which is inexpensive, without reagents specific to each species, in particular compared with the molecular biology methods, which gives a result in a short time, less than one hour, and which can be routinely used clinically, without requiring highly qualified personnel. Furthermore, the entire method for characterizing microorganisms can be advantageously carried out with the same mass spectrometer, thereby simplifying the instrumentation of the microbiological test laboratory.
To this end, the invention proposes a novel method for characterizing at least one microorganism from a sample, which comprises identifying said at least one microorganism and determining the properties of typing, potential resistance to at least one antimicrobial, and virulence factor, characterized in that the determining of the properties of typing, resistance to at least one antimicrobial, and virulence factor for said at least one microorganism is implemented by means of mass spectrometry using proteins, peptides and/or metabolites as markers of said properties of typing, resistance to at least one antimicrobial, and virulence factor.
Thus, the method of the invention is such that at least three of the properties for characterizing a microorganism are made use of by means of the mass spectrometry

3 technique using, as markers, proteins, peptides or metabolites representative of the microorganisms to be characterized.
The microorganisms that can be characterized by means of the method of the invention are all pathogenic or nonpathogenic microorganisms encountered both industrially and clinically. They may be bacteria, viruses, protozoa or yeasts.
The expression "markers of the properties of typing, resistance to at least one antimicrobial, and virulence factor" is intended to mean molecules, of protein or metabolic origin, which are characteristic of said properties.
The expression "typing a microorganism" is intended to mean the differentiation of several strains within the same species. Typing has an epidemiological value; the clinician knows whether the strain isolated from the patient comes from the same source as other strains that are apparently identical and isolated from other patients or from the environment. This thus makes it possible to reveal a seat of infection in a hospital or at the time of food poisoning. By way of nonlimiting examples of markers of typing properties in bacteria, mention may be made of peptides having characteristic mutations, such as the transcription products of the adk, fumC, gyrB, icd, mdh, pur A and recA genes of Escherichia coli, and those of the arc, aroE, glp¥, gmk, pta, tpi and yqiL genes of Staphylococcus aureus. By way of nonlimiting examples of markers of typing properties in protozoa, mention may be made of the products of the chitinase gene of Entamoeba histolytica and E. dispar. By way of nonlimiting examples of markers of typing properties in viruses, mention may be made of the products of the polymerase gene of the human immunodeficiency virus. Finally, by way of nonlimiting examples of markers of typing properties in yeasts, mention may be made of the products of transcription of the aatla, accl, adpl, mpih, syal, vps\3 and zwfih gene fragments of Candida albicans.
The expression "determining the resistance to at least one antimicrobial" is intended to mean determining a microorganism's susceptibility to being destroyed by an antimicrobial. Thus, if the microorganism is a bacterium, the antimicrobial against which it may develop a resistance is an antibiotic, if it is a protozoan, the antimicrobial is an antiparasitic, if it is a virus, the antimicrobial is an antiviral, and if it is a yeast, the antimicrobial is an antifungal. The proteins involved in the resistance mechanisms will differ according to the family and the species. By way of nonlimiting examples of markers of resistance to at least one antibiotic that are of use in bacteria, mention may

4 be made of the transcription products of the mecA gene of Staphylococcus aureus, conferring resistance to methicillin, and making it possible to indicate whether strains are methicillin-resistant (MRSA strains) or else methicillin-sensitive (MSSA strains). Mention may also be made of the TEM-2 protein which makes it possible to indicate whether Escherichia coli strains are resistant to penicillins but sensitive to other classes of antibiotics such as cephalosporins or carbapenems. Another marker is the enzyme called KPC (for Klebsiella pneumoniae carbapenemase) which confers resistance to carbapenems. Another example of a resistance marker for Staphylococcus aureus is the metabolic profile representative of the resistance to vancomycin as described by E. Alexander et al, in the poster "Metabolomics-based approach to antibiotic resistance in Staphylococcus aureus'' presented at the ASMS conference, 2009. By way of nonlimiting example of markers of resistance to at least one antiparasitic of use in protozoa, mention may be made of iron-containing superoxide dismutase (Fe-SOD) and peroxyredoxin, the increased expression of which confers resistance to metronidazole. By way of nonlimiting example of a marker of resistance to at least one antiviral of use in viruses, mention may be made of mutations of the human immunodeficiency virus reverse transcriptase enzyme, conferring decreased sensitivity to reverse-transcriptase nucleoside inhibitors. Finally, by way of nonlimiting example of markers of resistance to at least one antifungal of use in yeasts, mention may be made of the mutation of the Candida albicans 1,3-b-D-glucan synthase enzyme, which confers decreased sensitivity to echinocandins. For another example, mention may be made of resistance to azole antifungals in Candida albicans, in particular resistance to fluconazole. The target of fluconazole is an enzyme, lanosterol demethylase, involved in the synthesis of ergosterol, a main constituent of the fungal wall. The resistance to fluconazole may be associated with the appearance of point mutations in the erg 11 gene encoding lanosterol demethylase.
It should be noted that the resistance-specific markers can also be used as typing markers, as demonstrated by the applicant.
The expression "determining the virulence of a microorganism" is intended to mean evaluating the pathogenic, harmful and violent nature of the microorganism. By way of nonlimiting examples of a virulence marker in bacteria, mention may be made of PVL (Panton-Valentine Leukocidin), a cytolytic toxin with two synergistic components (LukFet LukS), present in Staphylococcus aureus, which is one of the

5 most virulent toxins causing skin conditions, extensive cellulitis, osteomyelitis and necrotizing pneumonia, and is involved in viral superinfections. Other examples comprise autolysin and pneumolysin present in Streptococcus pneumoniae, a species responsible for respiratory tract infections, meningitis and bacteriemia, and also toxins A and B of Clostridium difficile, a commensal bacterium of the intestine, which toxins either cause a modification of the permeability of the intestinal epithelium (toxin A), or directly attack the cells of the epithelium (toxin B), or decrease intestinal transit and intestinal absorption over time, causing diarrhoea (combined action of toxins A and B). Mention may also be made, as an example, of the Shiga toxins Stxl and Stx2 present in Escherichia coli. These two cytotoxins are considered to be important virulence factors of enterohemorrhagic Escherichia coli. They are responsible for complications such as ulcerative colitis or hemolytic-uremic syndrome. By way of nonlimiting example of a virulence marker in protozoa, mention may be made of antioxidants (Fe-hydrogenase 2, peroxiredoxin, superoxide dismutase) present in Entamoeba histolytica, a species responsible for dysentery and hepatic abscesses. By way of nonlimiting example of a virulence marker in viruses, mention may be made of the Nef protein variant in the human immunodeficiency virus type 1, the more pathogenic type in humans. Finally, by way of nonlimiting example of a virulence marker in yeasts, mention may be made of lipase 8 in Candida albicans, a species responsible for superficial candidiasis, but also septicemic and disseminated candidiasis.
It should be noted that the virulence-specific markers can also be used as a typing marker, as demonstrated by the applicant.
The method of the invention can be implemented for characterizing bacteria, said antimicrobial then being an antibiotic, which constitutes an embodiment of the invention. Thus, for example, by way of bacteria that can be characterized according to the method of the invention, mention may be made of:
-Escherichia coli using TEM-2 as resistance and typing marker, and also Shiga toxins, OmpA as virulence and typing marker.
- Enterococcus faecalis and faecium using VanA and VanB for resistance and typing, and also ESP (Enterococcal Surface Protein) for virulence and typing, or else
-Staphylococcus aureus using the protein known as Immunoglobulin G-binding protein A (also knovm as protein A) for typing, the PBP2a protein for resistance, or even typing, and also the PVL protein for virulence, or even also typing.

6
By way of other microorganisms that can be characterized according to the method of the invention, mention may be made of:
-Candida albicans using the 1,3-b-D-glucan synthase enzyme or else the lanosterol demethylase enzyme as resistance and typing marker, and also lipase 8 as virulence and typing marker.
The sample on which the method of the invention can be implemented is any sample capable of containing a target microorganism. The sample may be of biological origin, that is to say animal, vegetable or human origin. It may then correspond to a specimen of biological fluid (whole blood, serum, plasma, urine, cerebro-spinal fluid, organic secretion, for example), a tissue specimen or isolated cells. This specimen can be used as it is insofar as the markers for characterizing the microorganisms are available in the tested specimen, or else it can undergo, prior to the analysis, a preparation of enrichment, extraction, concentration, purification and/or culturing type, according to methods known to those skilled in the art.
The sample may be of industrial origin, i.e., according to a nonexhaustive list, a specimen of air, a specimen of water, a specimen taken from a surface, an object or a manufactured product, or a product of food origin. Among the samples of food origin, mention may be made, nonexhaustively, of a sample of a milk product (yoghurt, cheeses), of meat, of fish, of egg, of fruit, of a vegetable, of water or of a beverage (milk, fruit juice, soda, etc.). These samples of food origin may also come from sauces or prepared dishes. Finally, a food sample may be derived from an animal feed, such as in particular animal meals.
When the markers for characterizing microorganisms are of protein origin, upstream of the detection by mass spectrometry, the sample to be analyzed is preferentially pretreated in order to generate peptides from all the proteins present in the sample so as to fragment these proteins into peptides, for example by digestion with a proteolytic enzyme (protease), or via the action of a chemical reagent. Indeed, proteins can be cleaved by means of a physicochemical treatment, by means of a biological treatment or by means of a combination of the two treatments. Among the treatments that can be used, mention may be made of treatment with hydroxyl radicals, in particular with H2O2. Treatment with hydroxyl radicals causes cleavage of the peptide bonds, which takes place randomly on any peptide bond of the protein. The concentration of hydroxyl radicals conditions the number of cleavages made and

7 therefore the length of the peptide fragments obtained. Other chemical treatments can also be used, for instance treatment with cyanogen bromide (CNBr) which specifically breaks the peptide bonds at the level of the carboxyl group of methionyl residues. It is also possible to cany out a partial acid cleavage at the aspartyl residues by heating at 1000°C a solution of proteins in trifluoroacetic acid.
Treatment of the proteins by enzymatic digestion is nevertheless preferred compared with physicochemical treatments since it more extensively preserves the structure of the proteins, and is easier to control. The term "enzymatic digestion" is intended to mean the single or combined action of one or more enzymes under appropriate reaction conditions. The enzymes which carry out proteolysis, called proteases, cleave proteins at specific sites. Each protease generally recognizes a sequence of amino acids within which it always performs the same cleavage. Certain proteases recognize a single amino acid or a sequence of two amino acids between which they perform a cleavage, other proteases recognize only longer sequences. These proteases may be endoproteases or exoproteases. Among the known proteases, mention may be made, as described in WO2005/098071, of:
- specific enzymes, such as trypsin which splits the peptide bond at the level of the carboxylic group of Arg and Lys residues, endolysin which cleaves the peptide bond of the -CO group of lysines, chymotrypsin which hydrolyzes the peptide bond at the level of the carboxylic group of aromatic residues (Phe, Tyr and Trp), pepsin which cleaves at the level of the NH2 group of aromatic residues (Phe, Tyr and Trp), the V8 protease of the V8 strain of Staphylococcus aureus, which cleaves the peptide bond at the level of the carboxylic group of the Glu residue;
- nonspecific enzymes, such as thermolysin originating from the Bacillus thermoproteolyticus bacterium, which hydrolyzes the peptide bond of the NH2 group of hydrophobic amino acids (Xaa-Leu, Xaa-Ile, Xaa-Phe), subtilisin and pronase which are bacterial proteases that hydrolyze virtually all the bonds and can convert proteins into oligopeptides under controlled reaction conditions (enzyme concentration and reaction time).
Several proteases can be used simultaneously, if their methods of action are compatible, or they can be used successively. In the context of the invention, the digestion of the sample is preferably carried out via the action of a protease enzyme, for example trypsin.

8
The generation of peptides using a chemical reagent or a protease can be obtained by simple reaction in solution. It can also be carried out with a microwave oven [11], or under pressure [12], or alternatively with an ultrasonic device [13]. In the latter three cases, the protocol will be much faster.
Among the peptides thus obtained, the peptides specific for the protein are called proteotypic peptides. It is these which will be assayed by mass spectrometry.
According to one embodiment of the invention, the characterization markers are proteins of the microorganism to be characterized. In particular, said proteins are digested into peptides, preferably with an enzyme, more preferably with trypsin.
Similarly, the sample containing characterization markers of protein origin can also be pretreated for purification purposes. When the markers are of protein origin, this purification pretreatment can be carried out before or after the step of generating peptides as described above.
The sample purification pretreatment is widely known to those skilled in the art and may in particular implement centrifugation, filtration, electrophoresis or chromatography techniques. These separating techniques can be used alone or combined with one another in order to obtain a multidimensional separation. For example, a multidimensional chromatography can be used by combining a separation by ion exchange chromatography with a reverse-phase chromatography, as described by T. Fortin et al. [14], or H. Keshishian et al [15]. In these publications, the chromatographic medium may be in a column or a cartridge (solid-phase extraction).
The electrophoretic or chromatographic fraction (or the retention time in monodimensional or multidimensional chromatography) of the proteotypic peptides is characteristic of each peptide and the implementation of these techniques therefore makes it possible to select the proteotypic peptide(s) to be assayed. Such a fractionation of the peptides generated makes it possible to increase the specificity of the subsequent assay by mass spectrometry.
An alternative to electrophoresis or chromatography techniques, for the peptide fractionation, consists in specifically purifying the N-glycopeptides ([16] and patent application WO 2008/066629). Nevertheless, such a purification only allows the quantification of peptides having undergone a post-translation modification of N-glycosylation type. However, not all proteins are glycosylated, which therefore limits its use.

9
The mass spectrometry to be implemented in the method of the invention is widely known to those skilled in the art as a powerful tool for analyzing and detecting various types of molecules. Generally, any type of molecule that can be ionized can be detected as a function of its molecular weight using a mass spectrometer. Depending on the nature of the molecule to be detected, of protein or metabolic origin, certain mass spectrometry techniques may be more suitable. Nevertheless, whatever the mass spectrometry method used for the detection, the latter comprises a step of ionization of the target molecule into ions termed molecular ions, in the present case a step of ionization of the characterization markers, and a step of separation of the molecular ions obtained as a function of their weight.
All mass spectrometers therefore comprise:
i) an ionization source intended to ionize the markers present in the sample to be analyzed, i.e. to give these markers a positive or negative charge;
ii) a mass analyzer intended to separate the ionized markers, or molecular ions, according to their mass to charge ratio (m /z);
iii) a detector intended to measure the signal produced either directly by the molecular ions, or by ions produced from the molecular ions, as detailed hereinafter.
The ionization step necessary for implementing mass spectrometry can be carried out by any method known to those skilled in the art. The ionization source makes it possible to bring the molecules to be assayed into an ionized and gaseous state. An ionization source can be used either in positive mode for studying positive ions, or in negative mode for studying negative ions. Several types of sources exist and will be used depending on the desired result and the molecules analyzed. Mention may in particular be made of:
- electron ionization (EI), chemical ionization (CI) and desorption-chemical ionization (DCI),
- fast atom bombardment (FAB), metastable atom bombardment (MAB) or ion bombardment (SIMS, LSIMS),
- inductively coupled plasma (ICP),
- atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI),
- electrospray ionization (ESI),

10
- matrix assisted laser desorption iojaizatipn (MALDI), surface enhanced laser desorption ionization (SELDI) or desorption/ionization on silicon (DIOS),
- ionization-desorption by interaction with metastable species (DART).
In particular, the ionization can be carried out as follows: the sample containing the target molecules is introduced into an ionization source, where the molecules are ionized in the gas state and thus converted into molecular ions which correspond to the initial molecules. An ionization source of electrospray type (ESI for ElectroSpray lonisation) makes it possible to ionize a molecule while at the same time causing it to pass from a liquid state to a gas state. The molecular ions obtained then correspond to the molecules present in the liquid state, with, in the positive mode, one, two or even three additional protons or more, and therefore carry one, two or even three charges or more. For example, when the target molecule is a protein, ionization of the proteotypic peptides obtained after fractionation of the target protein, by virtue of a source of electrospray type operating in the positive mode, results in polypeptide ions in the gas state, with one, two or even three additional protons or more and which therefore carry one, two or even three charges or more, and allows change from a liquid state to a gas state [17]. This type of source is particularly suitable when the target molecules or proteotypic peptides obtained are separated beforehand by reverse-phase liquid chromatography. Nevertheless, the yield from ionization of the molecules present in the sample can vary according to the concentration and the nature of the various species present. This phenomenon results in a matrix effect knovra to those skilled in the art.
A MALDI ionization source will make it possible to ionize molecules from a sample in the solid state.
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 is any mass analyzer known to those skilled in the art. Mention may be made of low-resolution analyzers, of the quadrupole (Q), 3D ion trap (IT) or linear ion trap (LIT) type, also called ion trap, and high-resolution analyzers, for measuring the exact mass of the analytes and which use in particular the magnetic sector coupled to an electrical sector, the time of flight (TOF).
The separation of the molecular ions according to their m/z ratio can be implemented a single time (single mass spectrometry or MS), or else several successive

11
MS separations can be carried out. When two successive MS separations are carried out, the analysis is called MS/MS or MS^. When three successive MS separations are carried out, the analysis is called MS/MS/MS or MS^ and more generally, when n successive MS separations are carried out, the analysis is called MS".
Among the techniques implementing several successive separations, the SRM (Selected Reaction Monitoring) mode in the case of detection or assaying of a single target molecule, or else the MRM (Multiple Reaction Monitoring) mode in the case of detection or assaying of several target molecules, are particularly uses of MS separation. Similarly, the MRM'' mode is a particular use of separation by MS/MS/MS. The term targeted mass spectrometry is then used.
In the case of a detection in single MS mode, it is the mass/charge ratio of the molecular ions obtained which is correlated with the target molecule to be detected.
In the case of a detection in the MS/MS mode, essentially two steps are added, compared with an MS assay, which are:
i) a fragmentation of the molecular ions, then called precursor ions, to give ions termed Ist-generation fragment ions, and
ii) a separation of the ions termed Ist-generation fragment ions according to their mass (m/z)2, the ratio (m/z)i corresponding to the ratio (m/z) of the precursor ions.
It is then the mass/charge ratio of the Ist-generation fragment ions thus obtained which is correlated with the target molecule to be detected. The term "first-generation fragment ion" is intended to mean an ion resulting from the precursor ion, following a fragmentation step and the mass to charge ratio m/z of which is different than the precursor ion.
The pairs (m/z)i and (m/z)2 are named transitions and are representative of the characteristic ions to be detected.
The choice of the characteristic ions that are detected in order to be correlated with the target molecule is made by those skilled in the art according to standard methods. Their selection will advantageously result in assays which are as sensitive as possible, as specific as possible and as robust as possible, in terms of reproducibility and reliability. In the methods developed for the selection of proteotypic peptides (m/z)i, and of a first-generation fragment (m/z)2, the choice is essentially based on the intensity of the response. For further details, reference may be made to V. Fusaro et al. [18]. Commercial software, such as the MIDAS software and the MRM Pilot software

12 from Applied Biosystems or else MRMaid [19], may be used by those skilled in the art to allow them to predict all the possible transition pairs. Use may also be made of a database called PeptideAtlas, constructed by F. Desiere et al. [20] in order to compile all the peptide MRM transitions described by the scientific community. This PeptideAtlas database is freely available on the Internet. For nonprotein molecules, it is also possible to use databases, such as, for example, the one accessible through the Cliquid software from the company Applied Biosystems (United States of America).
An alternative approach for selecting the proteotypic peptides, (m/z)i and (m/z)2, consists in using the MS/MS fragmentation spectra obtained on the occasion of other studies. These studies may be, for example, the phases of discovery and identification of biomarkers by proteomic analysis. This approach was proposed by Thermo Scientific during a meeting of users [19]. It makes it possible to generate a hst of candidate transitions from the peptides identified experimentally by the SIEVE software (Thermo Scientific). Certain criteria have been detailed by J. Mead et al. [19] for the choice of the (m/z)i and (m/z)2 ions and are detailed hereinafter:
• Peptides with internal cleavage sites, i.e. with internal lysine or arginine, should be avoided, unless the lysine or the arginine is followed by proline.
• Peptides with asparagine or glutamine should be avoided because they can deaminate.
• Peptides with N-terminal glutamine or glutamic acid should be avoided because they can spontaneously cyclize.
• Peptides with methionine should be avoided because they can be oxidized.
• Peptides with cysteine should be avoided because they can be nonreproducibly modified during a possible step of denaturation, reduction and blocking of the thiol functions.
• Peptides with proline can be considered to be favorable because they generally produce intense firagments in MS/MS with a single very predominant peak. However, a single very predominant fragment does not make it possible to validate the identity of the transition in a complex mixture. Indeed, only the simultaneous presence of several characteristic fragments makes it possible to verify that the desired precursor ion is indeed detected.

13
• Peptides having a proline adjacent to the C-terminal (position n-1) or in the second position relative to the C-terminal (position n-2) are to be avoided because, in this case, the size of the first-generation fragment peptide is generally considered to be too small to be sufficiently specific.
• The selection of fragments having a mass greater than the precursor is to be preferred in order to promote the specificity. For this, it is necessary to select a doubly-charged precursor ion and to select the most intense first-generation fragment ion having a mass greater than the precursor, i.e. a singly-charged first-generation fragment ion.
The fragmentation of the precursor ions selected is carried out in a fragmentation cell such as the models of triple quadrupole type [21], or of ion trap type [22], or else of time of flight (TOP) type [23], which also make it possible to separate the ions. The fragmentation(s) will be conventionally carried out by collision with an inert gas such as argon or nitrogen, in an electrical field, by photoexcitation or photodissociation using an intense light source, collision with electrons or radical species, by application of a potential difference, for example in a time of flight tube, or by any other activation mode. The characteristics of the electrical field condition the intensity and the nature of the fragmentation. Thus, the electrical field applied in the presence of an inert gas, for example in a quadrupole, conditions the collision energy supplied to the ions. This collision energy will be optimized, by those skilled in the art, in order to increase the sensitivity of the transition to be assayed. By way of example, it is possible to vary the collision energy between 5 and 180 e'V in q2 in an AB SCIEX QTRAP® 5500 mass spectrometer from the company Applied Biosystems (Foster City, United States of America). Similarly, the duration of the collision step and the excitation energy within, for example, an ion trap will be optimized, by those skilled in the art, in order to produce the most sensitive assay. By way of example, it is possible to vary this duration, called excitation time, between 0.010 and 50 ms and the excitation energy between 0 and 1 (arbitrary unit) in Q3 in an AB SCIEX QTRAP® 5500 mass spectrometer from the company Applied Biosystems.
Finally, the detection of the characteristic ions selected is carried out conventionally, in particular by means of a detector and a processing system. The detector collects the ions and produces an electrical signal, the intensity of which depends on the amount of ions collected. The signal obtained is then amplified so that it

14 can be processed by computer. A data-processing computer assembly makes it possible to convert the information received by the detector into a mass spectrum.
The principle of the SRM mode, or else of the MRM mode, is to specifically select a precursor ion, to fragment it, and then to specifically select one of its fragment ions. For such applications, devices of the triple quadrupole type or triple quadrupole-ion trap hybrids are generally used.
In the case of a triple quadrupole device (Qlq2Q3) used in MS^ mode, for the purpose of assaying or detecting a target protein, the first quadrupole (Ql) makes it possible to filter the molecular ions, corresponding to the proteotypic peptides characteristic of the protein to be assayed and obtained during a prior digestion step, according to their mass to charge ratio (m/z). Only the peptides having the mass/charge ratio of the proteotypic peptide being sought, ratio called (m/z)i, are transmitted to the second quadrupole (q2) and play the role of precursor ions for the subsequent fragmentation. The q2 analyzer makes it possible to fragment the peptides of mass/charge ratio (m/z)i into first-generation fragment ions. The fragmentation is generally obtained by collision of the precursor peptides with an inert gas, for instance nitrogen or argon in q2. The first-generation fragment ions are transmitted to a third quadrupole (Q3) which filters the first-generation fragment ions according to a specific mass to charge ratio, which ratio is called (m/z)2. Only the first-generation fragment ions having the mass/charge ratio of a characteristic fragment of the proteotypic peptide sought (ni/z)2 are transmitted to the detector in order to be detected, or even quantified.
This operating mode has a double selectivity, in relation to the selection of, on the one hand, the precursor ion and the selection of, on the other hand, the first-generation fragment ion. Mass spectrometry in SRM or MRM mode is therefore advantageous for the quantification.
When the mass spectrometry implemented in the method of the invention is tandem mass spectrometry (MS^, MS^, MS'' or MS^), several mass analyzers can be coupled together. For example, a first analyzer separates the ions, a collision cell makes it possible to fragment the ions, and a second analyzer separates the fragment ions. Some analyzers, such as ion traps or FT-ICR, constitute several analyzers in one and make it possible to fragment the ions and to analyze the fragments directly.
According to preferred embodiments of the invention, the method of the invention comprises one or more of the following characteristics:

15
- the mass spectrometry, implemented for the properties of typing, potential resistance to at least one antimicrobial, and virulence factor, is a spectrometry of MS/MS type, which has the advantage of generating a fragment specific to the molecule to be detected or to be quantified, and thus of providing the assay method with great specificity;
- the MS/MS spectrometry is MRM, which has the advantage of using an analysis cycle time in the mass spectrometer of a few tens of milliseconds, which makes it possible to detect or quantify with great sensitivity, and in a multiplexed manner, a large number of different molecules;
- the determination of the properties of typing, resistance to an antimicrobial, and virulence factor is carried out in the same mass spectrometry apparatus, preferably simultaneously, which has the advantage of reducing the analysis time and the cost of the instrument; this also facilitates the processing and reporting of the results.
In addition to determining the properties of typing, resistance to an antimicrobial and virulence factor, it is advisable to identify the microorganism(s) present in the sample to be tested.
The methods for identifying microorganisms are widely knovm to those skilled in the art, as described, for example, by P. R. Murray el al, in Manual of Clinical Microbiology, 2007, 9th edition, and in particular in Vol. I, Section III, chapters 15 and 16 for bacteria and yeasts, Vol. II, Section VI, chapter 82 for viruses, and Vol. II, Section X, chapter 135 for protozoa. By way of example of conventional identification methods, mention may be made of the determination of the biological profile, using for example Vitek 2 identification cards (bioMerieux), or alternatively using molecular biology techniques with identification criteria based on studying the presence of certain genes, and on studying the sequence thereof.
The identification can be carried out directly from the sample in which the identification is performed, or else the microorganisms contained in the sample can be cultured by methods well known to those skilled in the art with culture media and optimum culture conditions adapted according to the species of microorganisms to be sought, as described by P.R. Murray et al, in Manual of Clinical Microbiology, 2007, 9th edition, Vol. I, Section III, chapter 14, in particular in Vol. I, Section IV, chapter 21 for bacteria, Vol. II, Section VI, chapter 81 for viruses, Vol. II, Section VIII, chapter 117 for yeasts, and Vol. II, Section X, chapter 134 for protozoa.

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Thus, in general, in the case of an identification, by means of a biochemical method, of a bacterium in a specimen, it is first necessary to obtain it in pure culture, for example after inoculation on agar. The molecular biology (PCR) can in certain cases be applied directly to the sample to be analyzed.
Instead of culturing the microorganisms, the latter can be concentrated by capture directly in the sample by means of active surfaces. Such a method has been described by W.-J. Chen et al. [11], who captured various bacterial species by means of magnetic beads with an Fe304/Ti02-activated surface. Capture by other means is also possible, such as capture by means of lectins [24], or by means of antibodies [25], or else by means of vancomycin [26]. The capture makes it possible to concentrate the microorganisms and thus to reduce or even eliminate the culture step. This results in a considerable time saving.
The identification can also be carried out by mass spectrometry, according to the techniques previously described, preferably by MS, by MS/MS, or else by MS followed by spectrometry of MS/MS type, which constitutes an embodiment of the invention. In this case also, the sample can be subjected beforehand to a culture step such as inoculation on agar.
The use of a method of identification by MS is advantageous since it can be carried out in a few minutes and it requires a mass spectrometer with a single analyzer, i.e. an instrument that is less complex than a tandem mass spectrometer used in MS/MS.
The use of a method of identification by MS followed by spectrometry of MS/MS type is also advantageous. It makes it possible to be sure of the identity of the ions observed in MS, which increases the specificity of the analysis.
The use of a method of identification by MS/MS of MRM type has the advantage of being more sensitive and simpler than the conventional approaches of MS then MS/MS. This method requires neither effective software for processing the information between the acquisition of the MS spectrum and of the MS/MS spectrum, nor any change in regulation of the machine parameters for linking MS and MS/MS spectra.
The method of identification by MS can be carried out with an electrospray source on a crude sample, as described by S. Vaidyanathan et al. [27] or else by R. Everley et al. [8] after chromatographic separation. Various ranges of m/z then make

17 it possible to identify the microorganisms. S. Vaidyanathan et al. have used a window between 200 and 2000 Th, and R. Everley et al. a window between 620 and 2450 Th. The mass spectra can also be deconvoluted in order to access the mass of the proteins independently of their charge state. R. Everley et al. have thus exploited the masses between approximately 5 000 and 50 000 Da. Alternatively, the method of identification by MS can also be carried out by means of MALDI-TOF, as described by Claydon et al. [3] and T. Krishnamurthy and P. Ross [4]. The analysis combines the acquisition of a mass spectrum and interpretation by expert software. It is extremely simple and can be performed in a few minutes. This method of identification is currently spreading through medical test laboratories [28].
The identification of bacteria by MS then MS/MS via their proteins present in the sample has been widely applied by many teams. By way of example, mention may be made of the recent studies by N. Manes et al. [29] who have studied the peptidome of Salmonella enterica, or the studies by R. Nandakumar et al. [30] or by L. Hemychova et al. [31] who have studied the proteome of bacteria after digestion of the proteins with trypsin. The conventional approach consists in i) acquiring an MS spectrum, ii) successively selecting each precursor ion observed on the MS spectrum with an intense signal, iii) successively fragmenting each precursor ion and acquiring its MS/MS spectrum, iv) searching protein databases such as SWISS-PROT or NCBI, through software such as Mascot (Matrix Science, London, United Kingdom) or SEQUEST (Thermo Scientific, Waltham, United States of America), in order to identify the peptide having a strong probability of corresponding to the MS/MS spectrum observed. This method can result in the identification of a microorganism if a protein or a peptide characteristic of the species is identified.
According to yet another embodiment, the identification of said at least one microorganism is carried out by means of a conventional identification method and the method of the invention comprises an additional step of confirming the identification of said at least one microorganism, which confirmation step is carried out by mass spectrometry, according to the techniques previously described for the identification of microorganisms.
According to one particular embodiment, the mass spectrometry of the confirmation step is mass spectrometry of MS/MS type, preferably an MRM.

18
One of the advantages of the use of mass spectrometry Hes in the fact that it is particularly useful to quantify molecules, in the present case the markers of the properties of typing and resistance to at least one antimicrobial. To do this, the current intensity detected, which is proportional to the amount of target molecule, is used. The current intensity thus measured may serve as a quantitative measurement for determining the amount of target molecule present, which is characterized by its expression in International System (SI) units of molW or kg/m'^ type, or by multiples or submultiples of these units, or by the usual derivatives of SI units, including multiples or submultiples thereof By way of nonlimiting example, units such as ng/ml or fmol/1 are units characterizing a quantitative measurement.
A calibration is nevertheless necessary in order to be able to correlate the area of the peak measured, corresponding to the intensity of current induced by the ions detected, with the amount of target molecule to be assayed. For this, the calibrations conventionally used in mass spectrometry may be implemented, in the context of the invention. MRM assays are conventionally calibrated using external standards or, preferably, using internal standards as described by T. Fortin et al. [14]. When the target molecule is a proteotypic peptide, making it possible to assay a protein of interest, the correlation between the quantitative measurement and the amount of target proteotypic peptide, and consequently of protein of interest, is obtained by calibrating the signal measured relative to a standard signal for which the amount to be assayed is known. The calibration can be carried out by means of a calibration curve, for example obtained by successive injections of standard proteotypic peptide at various concentrations (external calibration) or, preferentially, by internal calibration using a heavy peptide, as internal standard, for example in accordance with the AQUA, QconCAT or PSAQ methods detailed hereinafter. The term "heavy peptide" is intended to mean a peptide corresponding to the proteotypic peptide, but in which one or more carbon 12 (^^C) atoms is (are) replaced with carbon 13 ('^C), and/or one or more nitrogen 14 (''^N) atoms is (are) replaced with nitrogen 15 (^^N).
The use of heavy peptides, as internal standards (AQUA), has also been proposed in patent application US 2004/0229283. The principle is to artificially synthesize proteotypic peptides with amino acids comprising isotopes that are heavier than the usual natural isotopes. Such amino acids are obtained, for example, by replacing some of the carbon 12 ('^C) atoms with carbon 13 (^^C), or by replacing some

19 of the nitrogen 14 (^'^N) atoms with nitrogen 15 (^^N). The artificial peptide (AQUA) thus synthesized has rigorously the same physicochemical properties as the natural peptide (with the exception of a higher mass). It is generally added, at a given concentration, to the sample, upstream of the assay by mass spectrometry, for example between the treatment leading to the cleavage of the proteins of the sample of interest and the fractionation of the peptides obtained after the treatment step. As a result, the AQUA peptide is copurified with the natural peptide to be assayed, during the fractionation of the peptides. The two peptides are therefore injected simultaneously into the mass spectrometer, for the assay. They then undergo the same ionization yields in the source. Comparison of the areas of the peak for the natural and AQUA peptides, the concentration of which is known, makes it possible to calculate the concentration of the natural peptide and to thus work back to the concentration of the protein to be assayed. A variant of the AQUA technique has been proposed by J.-M. Pratt et al. [32] under the name QconCat. This variant is also described in patent application WO 2006/128492. It consists in concatenating various AQUA peptides and in producing the artificial polypeptide in the form of a heavy recombinant protein. The recombinant protein is synthesized with amino acids comprising heavy isotopes. In this way, it is possible to obtain a standard for calibrating the simultaneous assaying of several proteins at a lower cost. The QconCAT standard is added from the beginning, upstream of the treatment leading to the cleavage of the proteins and before the steps of protein fractionation, denaturation, reduction and then blocking of the thiol functions of the proteins, if these are present. The QconCAT standard therefore undergoes the same cycle of treatment leading to cleavage of the proteins as the natural protein, thereby making it possible to take into account the yield from the treatment step leading to the cleavage of the proteins. This is because the treatment, in particular by digestion, of the natural protein may not be complete. In this case, the use of an AQUA standard would result in the amount of natural protein being underestimated. For an absolute assay, it may therefore be important to take into account the yields from treatment leading to cleavage of the proteins. However, V. Brun et al. [33] have shown that, sometimes, the QconCAT standards do not reproduce exactly the yield from treatment, in particular by digestion, of the natural protein, doubtless owing to a different three-dimensional conformation of the QconCAT protein.

20 V. Brun et al. [33] have therefore proposed using a method called PSAQ which is described in patent application WO 2008/145763. In this case, the internal standard is a recombinant protein, which has the same sequence as the natural protein but has been synthesized with heavy amino acids. The synthesis is carried out ex-vivo with heavy amino acids. This standard has rigorously the same physicochemical properties as the natural protein (with the exception of a higher mass). It is added from the begiiming, before the protein fractionation step, when said step is present. It is therefore copurified with the native protein, during the protein fractionation step. It exhibits the same yield from treatment, in particular by digestion, as the native protein. The heavy peptide obtained after cleavage is also copurified with the natural peptide, if a peptide fractionation step is carried out. The two peptides are therefore injected simultaneously into the mass spectrometer, so as to be quantitatively assayed. They therefore undergo the same ionization yields in the source. Comparison of the areas of the peak of the natural peptides and of the reference peptides in the PSAQ method makes it possible to calculate the concentration of the protein to be assayed while taking into account all the steps of the assay method.
All these techniques, namely AQUA, QconCAT or PSAQ or any other calibration technique, used in assays by mass spectrometry and in particular in MRM or MS assays, may be implemented for carrying out the calibration, in the context of the invention.
According to one preferred embodiment, the method of the invention allows the characterization of^Staphylococcus aureus.
In particular, the characterization of Staphylococcus aureus uses at least one peptide as follows: 1. for the typing:
at least one peptide belonging to protein A having the following sequence SEQ ID
No. 1:
MKKKNIYSIRKLGVGIASVTLGTLLISGGVTPAANAAQHDEAQQNAFYQVL
NMPNLNADQRNGFIQSLKDDPSQSANVLGEAQKLNDSQAPKADAQQNKF
NKDQQSAFYEILNMPNLNEEQRNGFIQSLKDDPSQSTNVLGEAKKLNESQA
PKADNNFNKEQQNAFYEILNMPNLNEEQRNGFIQSLKDDPSQSANLLAEAK
KLNESQAPKADNKFNKEQQNAFYEILHLPNLNEEQRNGFIQSLKDDPSQSA
NLLAEAKKLNDAQAPKADNKFNKEQQNAFYEILHLPNLTEEQRNGFIQSLK

21 DDPSVSKEILAEAKKLNDAQAPKEEDNNKPGKEDGNKPGKEDGNKPGKED NKKPGKEDGNKPGKEDNKKPGKEDGNKPGKEDGNKPGKEDGNKPGKED GNKPGKEDGNGVHVVKPGDTVNDIAKANGTTADKIAADNKLADKNMIKP GQELVVDKKQPANHADANKAQALPETGEENPFIGTTVFGGLSLALGAALL AGRRREL
said peptides being chosen, preferably, from the peptides having the sequence SEQ ID No.2, 3,4, 5, 6, 7 and 8 as defined hereinafter:
Peptide SEQ ID No. Amino acid sequence Location in SEQ ID No. 1
2 DDPSQSANVLGEAQK 70-84
3 DQQSAFYEILNMPNLNEEQR 103-122
4 DDPSQSTNVLGEAK 131-144
5 EQQNAFYEILNMPNLNEEQR 161-180
6 DDPSQSANLLAEAK 189-202
7 DDPSVSK 305-311
8 lAADNK 437-442
2. For the potential resistance to at least one antibiotic
at least one peptide belonging to the PBP2a protein having the following sequence
SEQ ID No. 9:
MKKIKIVPLILIVVVVGFGIYFYASKDKEINNTIDAIEDKNFKQVYKDSSYIS
KSDNGEVEMTERPIKIYNSLGVKDINIQDRKIKKVSKNKKRVDAQYKIKTN
YGNIDRNVQFNFVKEDGMWKLDWDHSVIIPGMQKDQSIHIENLKSERGKIL
DRNNVELANTGTAYEIGIVPKNVSKKDYKAIAKELSISEDYIKQQMDQNWV
QDDTFVPLKTVKKMDEYLSDFAKKFHLTTNETESRNYPLEKATSHLLGYV
GPINSEELKQKEYKGYKDDAVIGKKGLEKLYDKKLQHEDGYRVTIVDDNS
NTIAHTLIEKKKKDGKDIQLTIDAKVQKSIYNNMKNDYGSGTAIHPQTGELL
ALVSTPSYDVYPFMYGMSNEEYNKLTEDKKEPLLNKFQITTSPGSTQKILTA
MIGLNNKTLDDKTSYKIDGKGWQKDKSWGGYNVTRYEVVNGNIDLKQAI
ESSDNIFFARVALELGSKKFEKGMKKLGVGEDIPSDYPFYNAQISNKNLDNE
ILLADSGYGQGEILINPVQILSIYSALENNGNINAPHLLKDTKNKVWKKNIIS
KENINLLTDGMQQVVNKTHKEDIYRSYANLIGKSGTAELKMKQGETGRQI
GWFISYDKDNPNMMMAINVKDVQDKGMASYNAKISGKVYDELYENGNK

22 KYDIDE
said peptides being chosen, preferably, from the peptides having the sequence SEQ ID No. 10 to 17 as defined hereinafter:
Peptide SEQ ID Amino acid sequence Location in SEQ ID No.9
No.
10 lYNSLGVK 69-76
il DINIQDR 77-83
12 ELSISEDYIK 189-198
13 FQITTSPGSTQK 395-406
14 ILTAMIGLNNK ' 407-417
15 YEVVNGNIDLK 446-456
16 VALELGSK 470-477
17 SYANLIGK 590-597
3. For the virulence:
at least one peptide belonging to the PVL protein, subunits LukS and LukF, having
the following sequences SEQ ID No. 18 and 22, respectively:
SEQ ID No. 18:
MIFMVKKRLLAATLSLGIITPIATSFHESKADNNIENIGDGAEVVKRTEDTSS
DKWGVTQNIQVDFVKDKKYNKDALILKMQGFINSKTTYYNYKNTDHIKA
MRWPFQYNIGLKTNDPNVDLINYLPKNKIDSVNVSQTLGYNIGGNFNSGPS
TGGNGSFNYSKTISYNQQNYISEVERQNSKSVQWGIKANSFITSLGKMSGH
DPNLFVGYKPYSQNPRDYFVPDNELPPLVHSGFNPSFIATVSHEKGSGDTSE
FEITYGRNMDVTHATRRTTHYGNSYLEGSRIHNAFVNRNYTVKYEVNWKT
HEIKVKGHN
SEQ ID No. 22:
MKKIVKSSVVTSIALLLLSNTVDAAQHITPVSEKKVDDKITLYKTTATSDSD
KLKISQILTFNFIKDKSYDKDTLILKAAGNIYSGYTKPNPKDTISSQFYWGSK
YNISINSDSNDSVNVVDYAPKNQNEEFQVQQTVGYSYGGDINISNGLSGGG
NGSKSFSETINYKQESYRTSLDKRTNFKKIGWDVEAHKIMNNGWGPYGRD
SYHSTYGNEMFLGSRQSNLNAGQNFLEYHKMPVLSRGNFNPEFIGVLSRKQ

23 NAAKKSKITVTYQREMDRYTNFWNQLHWIGNNYKDENRATHTSIYEVDW ENHTVKLIDTQSKEKNPMS
said peptides being chosen, preferably, from the peptides having the sequences SEQ ID No. 19, 20, 21, 23 and 24 as defined hereinafter:
Peptide SEQ ID Amino acid sequence Location in SEQ ID No. 18
No.
19 TNDPNVDLINYLPK 115-128
20 SVQWGIK 184-190
21 ANSFITSLGK 191-200
Peptide SEQ ID Amino acid sequence Location in SEQ ID No.22
No.
23 MPVLSR 237-242
24 GNFNPEFIGVLSR 243-255
4. For the identification:
at least one peptide belonging to the SOS ribosomal protein L30 (RL30), SOS ribosomal protein L331 (RL331), staphylococcal secretory antigen ssaA2 (SSAA2), UPF0337 protein SA0772 (Y772), biftinctional autolysin (ATL), elongation factor Tu (EFTU), probable transglycolase isaA (ISAA) and UPF0457 protein SAl975.1 (Y197A), having the following sequences SEQ ID No. 25, 28, 31, 33, 36, 39, 41 and 43, respectively: SEQ ID No. 25:
MAKLQITLTRSVIGRPETQRKTVEALGLKKTNSSVVVEDNPAIRGQINKVK HLVTVEEK
SEQ ID No. 28: MRVNVTLACTECGDRNYITTKNKRNNPERVEMKKFCSRENKQTLHRETK
SEQ ID No. 31:
MKKIATATIATAGFATIAIASGNQAHASEQDNYGYNPNDPTSYSYTYTIDA QGNYHYTWKGNWHPSQLNQDNGYYSYYYYNGYNNYNNYNNGYSYNNY SRYNNYSNNNQSYNYNNYNSYNTNSYRTGGLGASYSTSSNNVQVTTTMA

24 PSSNGRSISSGYTSGRNLYTSGQCTYYVFDRVGGKIGSTWGNASNWANAA ARAGYTVNNTPKAGAIMQTTQGAYGHVAYVESVNSNGSVRVSEMNYGY GPGVVTSRTISASQAAGYNFIH
SEQIDNo. 33:
MADESKFEQAKGNVKETVGNVTDNKNLENEGKEDKASGKAKEFVENAKE
KATDFIDKVKGNKGE
SEQ ID No. 36:
MAKKFNYKLPSMVALTLVGSAVTAHQVQAAETTQDQTTNKNVLDSNKVK
ATTEQAKAEVKNPTQNISGTQVYQDPAIVQPKTANNKTGNAQVSQKVDTA
QVNGDTRANQSATTNNTQPVAKSTSTTAPKTNTNVTNAGYSLVDDEDDNS
EHQINPELIKSAAKPAALETQYKAAAPKAKTEATPKVTTFSASAQPRSVAA
TPKTSLPKYKPQVNSSINDYIRKNNLKAPKIEEDYTSYFPKYAYRNGVGRPE
GIVVHDTANDRSTINGEISYMKNNYQNAFVHAFVDGDRIIETAPTDYLSWG
VGAVGNPRFINVEIVHTHDYASFARSMNNYADYAATQLQYYGLKPDSAEY
DGNGTVWTHYAVSKYLGGTDHADPHGYLRSHNYSYDQLYDLINEKYLIK
MGKVAPWGTQFTTTPTTPSKPTTPSKPSTGKLTVAANNGVAQIKPTNSGLY
TTVYDKTGKATNEVQKTFAVSKTATLGNQKFYLVQDYNSGNKFGWVKEG
DVVYNTAKSPVNVNQSYSIKSGTKLYTVPWGTSKQVAGSVSGSGNQTFKA
SKQQQIDKSIYLYGSVNGKSGWVSKAYLVDTAKPTPTPIPKPSTPTTNNKLT
VSSLNGVAQINAKNNGLFTTVYDKTGKPTKEVQKTFAVTKEASLGGNKFY
LVKDYNSPTLIGWVKQGDVIYNNAKSPVNVMQTYTVKPGTKLYSVPWGT
YKQEAGAVSGTGNQTFKATKQQQIDKSIYLFGTVNGKSGWVSKAYLAVPA
APKKAVAQPKTAVKAYTVTKPQTTQTVSKIAQVKPNNTGIRASVYEKTAK
NGAKYADRTFYVTKERAHGNETYVLLNNTSHNIPLGWFNVKDLNVQNLG
KEVKTTQKYTVNKSNNGLSMVPWGTKNQVILTGNNIAQGTFNATKQVSV
GKDVYLYGTINNRTGWVNAKDLTAPTAVKPTTSAAKDYNYTYVIKNGNG
YYYVTPNSDTAKYSLKAFNEQPFAVVKEQVINGQTWYYGKLSNGKLAWI
KSTDLAKELIKYNQTGMTLNQVAQIQAGLQYKPQVQRVPGKWTDANFND
VKHAMDTKRLAQDPALKYQFLRLDQPQNISIDKINQFLKGKGVLENQGAA
FNKAAQMYGINEVYLISHALLETGNGTSQLAKGADVVNNKVVTNSNTKY
HNVFGIAAYDNDPLREGIKYAKQAGWDTVSKAIVGGAKFIGNSYVKAGQN

25 TLYKMRWNPAHPGTHQYATDVDWANINAKIIKGYYDKIGEVGKYFDIPQY K
SEQ ID No. 39:
MAKEKFDRSKEHANIGTIGHVDHGKTTLTAAIATVLAKNGDSVAQSYDMI
DNAPEEKERGITINTSHIEYQTDKRHYAHVDCPGHADYVKNMITGAAQMD
GGILVVSAADGPMPQTREHILLSRNVGVPALVVFLNKVDMVDDEELLELV
EMEVRDLLSEYDFPGDDVPVIAGSALKALEGDAQYEEKILELMEAVDTYIP
TPERDSDKPFMMPVEDVFSITGRGTVATGRVERGQIKVGEEVEIIGLHDTSK
TTVTGVEMFRKLLDYAEAGDNIGALLRGVAREDVQRGQVLAAPGSITPHT
EFKAEVYVLSKDEGGRHTPFFSNYRPQFYFRTTDVTGVVHLPEGTEMVMP
GDNVEMTVELIAPIAIEDGTRFSIREGGRTVGSGVVTEIIK
SEQ ID No. 41:
^4KKTIMASSLAVALGVTGYAAGTGHQAHAAEVNVDQAHLVDLAHNHQD
QLNAAPIKDGAYDIHFVKDGFQYNFTSNGTTWSWSYEAANGQTAGFSNVA
GADYTTSYNQGSNVQSVSYNAQSSNSNVEAVSAPTYHNYSTSTTSSSVRLS
NGNTAGATGSSAAQIMAQRTGVSASTWAAIIARESNGQVNAYNPSGASGL
FQTMPGWGPTNTVDQQINAAVKAYKAQGLGAWGF
SEQ ID No. 43:
MAMTVKKDNNEVRIQWRVADIKIPTSEIKNITQDQDIHAVPKLDSKDVSRI
GSTFGKTNRVIIDTEDHEYIIYTQNDQKVYNELTK
said peptides being chosen, preferably, from the peptides having the sequences SEQ ID No.26, 27, 29, 30, 32, 34, 35, 37, 38, 40,42,44 and 45 as defined hereinafter:
Peptide SEQ ID No. Amino acid sequence Location in SEQ ID No.25
26 LQITLTR 4^10
27 TNSSVWEDNPAIR 31-34
Peptide SEQ ID No. Amino acid sequence Location in SEQ ID No.28
29 VNVTLACTECGDR 3^05
30 NYITTK 16^21

26
Peptide SEQ JD No. Amino acid sequence Location in SEQ ID No.31
32 AGYTVNNTPK 200-209
Peptide SEQ ID No. Amino acid sequence Location in SEQ ID No.33
34 EFVENAKEK 42-50
35 ATDFIDKVK 51-59
Peptide SEQ ID No. Amino acid sequence Location in SEQ ID No.36
37 LYSVPWGTYK 696-705
38 AYLAVPAAPK 747-756
Peptide SEQ ID No. Amino acid sequence Location in SEQ ID No.39
40 TVGSGWTEinC 383-394
Peptide SEQ ID No. Amino acid sequence Location in SEQ ID No.41
42 LSNGNTAGATGSSAAQ- 148-168
MAQR
Peptide SEQ ID No. Amino acid sequence Location in SEQ ID No.43
44 NITQDQDIHAVPK 30-42
45 LDSKDVSR 43-50
It should be noted, as previously indicated, that, for the typing, the method of the invention can also use at least one peptide having the sequence SEQ ID No.: 10, 11, 12, 13, 14, 15, 16, 17, 19, 20, 21, 23, 24, 26, 27, 29, 30, 32, 34, 35, 37, 38, 40, 42, 44 and 45, which are of use for determining the potential resistance to at least one antibiotic, or the virulence, as previously indicated.
Of course, the term "at least one peptide" is intended to mean at least one, at least two, at least three, at least four, at least five, at least six or more peptides representative of the marker that it is desired to detect. Preferably, at least two, or even at least three, or even at least four, peptides per characteristic will be used.
According to another preferred embodiment, the method of the invention allows the characterization of Escherichia coli.
In particular, the characterization oi Escherichia coli uses at least one peptide as follows:
1. for the typing:
at least one peptide belonging to the aspartate ammonia-lyase protein (ASPA), the
ATP synthase alpha subunit (ATPA), 10 kDa chaperonin (CHIO), 60 kDa

27 chaperonin (CH60), the DNA-binding protein HU-beta (DBHB), glutamate decarboxylase (DCEB), succinate dehydrogenase flavoprotein subunit (DHSA), the DNA protection during starvation protein (DPS), the DNA-binding protein H-NS (HNS), malate dehydrogenase (MDH), phosphoglycerate kinase (PGK), phosphoribosylaminoimidazole-succinocarboxamide synthase (PUR7), the SOS ribosomal protein L4 (RL4), the 30S ribosomal protein SI (RSI), the UPF0076 protein yjgF (YJGF), having the following sequences SEQ ID No. 138 to 152, respectively:
SEQ ID No. 138
MSNNIRIEEDLLGTREVPADAYYGVHTLRAIENFYISNNKISDIPEFVRGMV
MVKKAAAMANKELQTIPKSVANAIIAACDEVLNNGKCMDQFPVDVYQGG
AGTSVNMNTNEVLANIGLELMGHQKGEYQYLNPNDHVNKCQSTNDAYPT
GFRIAVYSSLIKLVDAINQLREGFERKAVEFQDILKMGRTQLQDAVPMTLG
QEFRAFSILLKEEVKNIQRTAELLLEVNLGATAIGTGLNTPKEYSPLAVKKL
AEVTGFPCVPAEDLIEATSDCGAYVMVHGALKRLAVKMSKICNDLRLLSS
GPRAGLNEINLPELQAGSSIMPAKVNPVVPEVVNQVCFKVIGNDTTVTMAA
EAGQLQLNVMEPVIGQAMFESVHILTNACYNLLEKCINGITANKEVCEGYV
YNSIGIVTYLNPFIGHHNGDIVGKICAETGKSVREVVLERGLLTEAELDDIFS
VQNLMHPAYKAKRYTDESEQ
SEQ ID No. 139
MQLNSTEISELIKQRIAQFNVVSEAHNEGTIVSVSDGVIRIHGLADCMQGEM
ISLPGNRYAIALNLERDSVGAVVMGPYADLAEGMKVKCTGRILEVPVGRG
LLGRVVNTLGAPIDGKGPLDHDGFSAVEAIAPGVIERQSVDQPVQTGYKAV
DSMIPIGRGQRELIIGDRQTGKTALAIDAIINQRDSGIKCIYVAIGQKASTISN
VVRKLEEHGALANTIVVVATASESAALQYLAPYAGCAMGEYFRDRGEDAL
IIYDDLSKQAVAYRQISLLLRRPPGREAFPGDVFYLHSRLLERAARVNAEYV
EAFTKGEVKGKTGSLTALPIIETQAGDVSAFVPTNVISITDGQIFLETNLFNA
GIRPAVNPGISVSRVGGAAQTKIMKKLSGGIRTALAQYRELAAFSQFASDLD
DATRKQLDHGQKVTELLKQKQYAPMSVAQQSLVLFAAERGYLADVELSKI
GSFEAALLAYVDRDHAPLMQEINQTGGYNDEIEGKLKGILDSFKATQSW

28
SEQIDNo. 140
MNIRPLHDRVIVKRKEVETKSAGGIVLTGSAAAKSTRGEVLAVGNGRILEN
GEVKPLDVKVGDIVIFNDGYGVKSEKIDNEEVLIMSESDILAIVEA
SEQ ID No. 141
MAAKDVKFGNDARVKMLRGVNVLADAVKVTLGPKGRNVVLDKSFGAPTI
TKDGVSVAREIELEDKFENMGAQMVKEVASKANDAAGDGTTTATVLAQA
IITEGLKAVAAGMNPMDLKRGIDKAVTAAVEELKALSVPCSDSKAIAQVGT
ISANSDETVGKLIAEAMDKVGKEGVITVEDGTGLQDELDVVEGMQFDRGY
LSPYFINKPETGAVELESPFILLADKKISNIREMLPVLEAVAKAGKPLLIIAED
VEGEALATLVVNTMRGFVKVAAVKAPGFGDRRKAMLQDIATLTGGTVISE
EIGMELEKATLEDLGQAKRVVINKDTTTIIDGVGEEAAIQGRVAQIRQQIEE
ATSDYDREKLQERVAKLAGGVAVIKVGAATEVEMKEKKARVEDALHATR
AAVEEGVVAGGGVALIRVASKLADLRGQNEDQNVGIKVALRAMEAPLRQI
VLNCGEEPSVVANTVKGGDGNYGYNAATEEYGNMIDMGILDPTKVTRSAL
QYAASVAGLMITTECMVTDLPKNDAADLGAAGGMGGMGGMGGMM
SEQIDNo. 142
MNKSQLIDKIAAGADISKAAAGRALDAIIASVTESLKEGDDVALVGFGTFA
VKERAARTGRNPQTGKEITIAAAKVPSFRAGKALKDAVN
SEQ ID No. 143
MDKKQVTDLRSELLDSRFGAKSISTIAESKRFPLHEMRDDVAFQIINDELYL
DGNARQNLATFCQTWDDENVHKLMDLSINKNWIDiCEEYPQSAAIDLRCVN
MVADLWHAPAPKNGQAVGTNTIGSSEACMLGGMAMKWRWRKRMEAAG
KPTDKPNLVCGPVQICWHKFARYWDVELREIPMRPGQLFMDPKRMIEACD
ENTIGVVPTFGVTYTGNYEFPQPLHDALDKFQADTGIDIDMHIDAASGGFL
APFVAPDIVWDFRLPRVKSISASGHKFGLAPLGCGWVIWRDEEALPQELVF
NVDYLGGQIGTFAINFSRPAGQVIAQYYEFLRLGREGYTKVQNASYQVAA
YLADEIAKLGPYEFICTGRPDEGIPAVCFKLKDGEDPGYTLYDLSERLRLRG
WQVPAFTLGGEATDIVVMRIMCRRGFEMDFAELLLEDYKASLKYLSDHPK
LQGIAQQNSFKHT

29 SEQ ID No. 144
MKLPVREFDAVVIGAGGAGMRAALQISQSGQTCALLSKVFPTRSHTVSAQ , GGITVALGNTHEDNWEWHMYDTVKGSDYIGDQDAIEYMCKTGPEAILELE HMGLPFSRLDDGRIYQRPFGGQSKNFGGEQAARTAAAADRTGHALLHTLY QQNLKNHTTIFSEWYALDLVKNQDGAVVGCTALCIETGEVVYFKARATVL ATGGAGRIYQSTTNAHINTGDGVGMAIRAGVPVQDMEMWQFHPTGIAGA GVLVTEGCRGEGGYLLNKHGERFMERYAPNAKDLAGRDVVARSIMIEIRE GRGCDGPWGPHAKLKLDHLGKEVLESRLPGILELSRTFAHVDPVKEPIPVIP TCHYMMGGIPTKVTGQALTVNEKGEDVVVPGLFAVGEIACVSVHGANRL GGNSLLDLVVFGRAAGLHLQESIAEQGALRDASESDVEASLDRLNRWNNN RNGEDPVAIRKALQECMQHNFSVFREGDAMAKGLEQLKVIRERLKNARLD DTSSEFNTQRVECLELDNLMETAYATAVSANFRTESRGAHSRFDFPDRDDE NWLCHSLYLPESESMTRRSVNMEPKLRPAFPPKIRTY
SEQ ID No. 145
MSTAKLVKSKATNLLYTRNDVSDSEKKATVELLNRQVIQFIDLSLITKQAH
WNMRGANFIAVHEMLDGFRTALIDHLDTMAERAVQLGGVALGTTQVINS
KTPLKSYPLDIHNVQDHLKELADRYAIVANDVRKAIGEAKDDDTADILTAA
SRDLDKFLWFIESNIE
SEQ ID No. 146
MSEALKILNNIRTLRAQARECTLETLEEMLEKLEVVVNERREEESAAAAEV EERTRKLQQYREMLIADGIDPNELLNSLAAVKSGTKAKRAQRPAKYSYVD ENGETKTWTGQGRTPAVIKKAMDEQGKSLDDFLIKQ
SEQ ID No. 147
MKVAVLGAAGGIGQALALLLKTQLPSGSELSLYDIAPVTPGVAVDLSHIPT
AVKIKGFSGEDATPALEGANVVLISAGVARKPGMDRSDLFNVNAGIVKNL
VQQVAKTCPKACIGIITNPVNTTVAIAAEVLKKAGVYDKNKLFGVTTLDIIR
SNTFVAELKGKQPGEVEVPVIGGHSGVTILPLLSQVPGVSFTEQEVADLTKR
IQNAGTEVVEAKAGGGSATLSMGQAAARFGLSLVRALQGEQGVVECAYV
EGDGQYARFFSQPLLLGKNGVEERKSIGTLSAFEKNALEGMLDTLKKDIAL
GEEFVNK

30
SEQ ID No. 148
MSVIKMTDLDLAGKRVFIR7\DLNVPVKDGKVTSDARIRASLPTIELALKQG
AKVMVTSHLGRPTEGEYNEEFSLLPVVNYLKDKLSNPVRLVKDYLDGVDV
AEGELVVLENVRFNKGEKKDDETLSKKYAALCDVFVMDAFGTAHRAQAS
THGIGKFADVACAGPLLAAELDALGKALKEPARPMVAIVGGSKVSTKLTV
LDSLSKIADQLIVGGGIANTFIAAQGHDVGKSLYEADLVDEAKRLLSTCNIP
VPSDVRVATEFSETAPATLKSVNDVKADEQILDIGDASAQELAEILKNAKTI
LWNGPVGVFEFPNFRKGTEIVANAIADSEAFSIAGGGDTLAAIDLFGIADKIS
YISTGGGAFLEFVEGKVLPAVAMLEERAKK
SEQ ID No. 149
MQKQAELYRGKAKTVYSTENPDLLVLEFRNDTSAGDGARIEQFDRKGMV
NNKFNYFIMSKLAEAGIPTQMERLLSDTECLVKKLDMVPVECVVRNRAAG
SLVKRLGIEEGIELNPPLFDLFLKNDAMHDPMVNESYCETFGWVSKENLAR
MKELTYKANDVLKKLFDDAGLILVDFKLEFGLYKGEVVLGDEFSPDGSRL
WDKETLEKMDKDRFRQSLGGLIEAYEAVARRLGVQLD
SEQ ID No. 150
MELVLKDAQSALTVSETTFGRDFNEALVHQVVVAYAAGARQGTRAQKTR
AEVTGSGKKPWRQKGTGRARSGSIKSPIWRSGGVTFAARPQDHSQKVNKK
MYRGALKSILSELVRQDRLIVVEKFSVEAPKTKLLAQKLKDMALEDVLIITG
ELDENLFLAARNLHKVDVRDATGIDPVSLIAFDKVVMTADAVKQVEEMLA
SEQ ID No. 151
MTESFAQLFEESLKEIETRPGSIVRGVVVAIDKDVVLVDAGLKSESAIPAEQF
KNAQGELEIQVGDEVDVALDAVEDGFGETLLSREKAKRHEAWITLEKAYE
DAETVTGVINGKVKGGFTVELNGIRAFLPGSLVDVRPVRDTLHLEGKELEF
KVIKLDQKRNNVVVSRRAVIESENSAERDQLLENLQEGMEVKGIVKNLTD
YGAFVDLGGVDGLLHITDMAWKRVKHPSEIVNVGDEITVKVLKFDRERTR
VSLGLKQLGEDPWVAIAKRYPEGTKLTGRVTNLTDYGCFVEIEEGVEGLVH
VSEMDWTNKNIHPSKVVNVGDVVEVMVLDIDEERRRISLGLKQCKANPW
QQFAETHNKGDRVEGKIKSITDFGIFIGLDGGIDGLVHLSDISWNVAGEEAV

31 REYKKGDEIAAVVLQVDAERERISLGVKQLAEDPFNNWVALNKKGAIVTG KVTAVDAKGATVELADGVEGYLRASEASRDRVEDATLVLSVGDEVEAKF TGVDRKNRAISLSVRAKDEADEKDAIATVNKQEDANFSNNAMAEAFKAA KGE
SEQIDNo. 152
MSKTIATENAPAAIGPYVQGVDLGNMIITSGQIPVNPKTGEVPADVAAQAR QSLDNVKAIVEAAGLKVGDIVKTTVFVKDLNDFATVNATYEAFFTEHNAT FPARSCVEVARLPKDVKIEIEAIAVRR
said peptides being chosen, preferably, from the peptides having the sequence SEQ ID No.67 to 84 as defined hereinafter:
Peptide SEQ ID No. Amino acid sequence Location in SEQ ID No. 138
67 r ISDIPEFVR 41.49
68 lEEDLLGTR ^s
69 LVDAINQLR 163^171
Peptide SEQ ID No. Amino acid sequence Location in SEQ ID No. 139
70 TALAIDAIINQR 176^187
71 WNTLGAPIDGK loTTlS Peptide SEQ ID No. Amino acid sequence Location in SEQ ID No. 140
72 SAGOrVLTGSAAAK 21-34 Peptide SEQ ID No. Amino acid sequence Location in SEQ ID No. 141
73 AVTAAVEELK 123^132 Peptide SEQ ID No. Amino acid sequence Location in SEQ ID No. 142
74 ALDAIIASVTESLK 24-37 Peptide SEQ ID No. Amino acid sequence Location in SEQ ID No. 143
75 YWDVELR 17^178 Peptide SEQ ID No. Amino acid sequence Location in SEQ ED No. 144
76 LPGILELSR 327.335 Peptide SEQ ID No. Amino acid sequence Location in SEQ ID No. 145
77 SKATNLLYTR ^Tig
Peptide SEQ ID No. Amino acid sequence Location in SEQ ID No. 146
78 SEALKILNNIR '^

32
Peptide SEQ ID No. Amino acid sequence Location in SEQ ID No. 147
79 LFGVTTLDIIR 145-155 Peptide SEQ ID No. Amino acid sequence Location in SEQ ID No. 148
80 ASLPTIELALK 39-49
Peptide SEQ ID No. Amino acid sequence Location in SEQ ID No. 149
81 LLSDTECLVK 73-82
Peptide SEQ ID No. Amino acid sequence Location in SEQ ID No. 150
82 SILSELVR 107-114 Peptide SEQ ID No. Amino acid sequence Location in SEQ ID No. 151
83 GGFTVELNGIR 118-128 Peptide SEQ ID No. Amino acid sequence Location in SEQ ID No. 152
84 TGEVPADVAAQAR 39-51
2. For the potential resistance to at least one antibiotic
at least one peptide belonging to the TEM-2 beta-lactamase protein (TEM-2)
having the following sequence SEQ ID No. 126:
HPETLVKVKDAEDKLGARVGYIELDLNSGKILESFRPEERFPMMSTFKVLL CGAVLSRVDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAA ITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDE RDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPA GWFIADKSGVGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAE IGASLIKHW
said peptides being chosen, preferably, from the peptides having the sequence SEQ ID No.62 to 66 as defined hereinafter:
Peptide SEQ ID No. Amino acid sequence Location in SEQ ED No. 126
62 LLTGELLTLASR 168-179
63 SALPAGWFIADK 198-209
64 VAGPLLR 191-197
65 VGYIELDLNSGK 19-30
66 VLLCGAVLSR 49-58

33
3. For the virulence:
at least one peptide belonging to the Shiga toxin 1 subunit A protein (STXl A), the Shiga toxin 2 subunit A protein (STX2A) or to both, having the sequences SEQ ID No. 153 and 154, respectively:
SEQ ID No. 153
MKIIIFRVLTFFFVIFSVNVVAKEFTLDFSTAKTYVDSLNVIRSAIGTPLQTISS
GGTSLLMIDSGTGDNLFAVDVRGIDPEEGRFNNLRLIVERNNLYVTGFVNR
TNNVFYRFADFSHVTFPGTTAVTLSGDSSYTTLQRVAGISRTGMQINRHSLT
TSYLDLMSHSGTSLTQSVARAMLRFVTVTAEALRFRQIQRGFRTTLDDLSG
RSYVMTAEDVDLTLNWGRLSSVLPDYHGQDSVRVGRISFGSINAILGSVALI
LNCHHHASRVARMASDEFPSMCPADGRVRGITHNKILWDSSTLGAILMRR
TISS
SEQ ID No. 154
MKCILFKWVLCLLLGFSSVSHSREFTIDFSTQQSYVSSLNSIRTEISTPLEHIS QGTTSVSVINHTPPGSYFAVDIRGLDVYQARFDHLRLIIEQNNLYVAGFVNT ATNTFYRFSDFTHISVPGVTTVSMTTDSSYTTLQRVAALERSGMQISRHSLV SSYLALVEFSGNTMTRDASRAVLRFVTVTAEALRFRQIQREFRQALSETAP VYTMTPGDVDLTLNWGRISNVLPEYRGEDGVRVGRISFNNISAILGTVAVIL NCHHQGARSVRAVNEDSQPECQITGDRPVIKINNTLWESNTAAAFLNRKSQ FLYTTGK
said peptides being chosen, preferably, from the peptides having the sequences SEQ ID No.85, 86 and 87 as defined hereinafter:
Peptide SEQ ID No. Amino acid sequence Location in SEQ ED No. 153
85 TYVDSLNVIR 34.43
86 FVTVTAEALR 183^192
Peptide SEQ ID No. Amino acid sequence Location in SEQ ID No. 154
86 FVTVTAEALR 183^492
87 ISNVLPEYR 227-235

34 4. For the identification:
at least one peptide belonging to aconitate hydratase 2 (AC0N2), L-asparaginase 2 (ASPG2), 3-oxoacyl-[acyl-carrier-protein] synthase 1 (FABB), glutamine-binding periplasmic protein (GLNH), molybdate-binding periplasmic protein (MODA), dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex (0DP2), outer membrane protein C (OMPC), formate acetyltransferase 1 (PFLB), succinyl-CoA ligase [ADP-forming] subunit alpha (SUCD), transketolase 1 (TKTl), UPF0381 protein yfcZ (YFCZ), uncharacterized protein ygaU (YGAU), having the following sequences SEQ ID No. 127 to 135,176,136 and 137, respectively:
SEQ ID No. 127
MLEEYRKHVAERAAEGIAPKPLDANQMAALVELLKNPPAGEEEFLLDLLT
NRVPPGVDEAAYVKAGFLAAIAKGEAKSPLLTPEKAIELLGTMQGGYNIHP
LIDALDDAKLAPIAAKALSHTLLMFDNFYDVEEKAKAGNEYAKQVMQSW
ADAEWFLNRPALAEKLTVTVFKVTGETNTDDLSPAPDAWSRPDIPLHALA
MLKNAREGIEPDQPGVVGPIKQIEALQQKGFPLAYVGDVVGTGSSRKSATN
SVLWFMGDDIPHVPNKRGGGLCLGGKIAPIFFNTMEDAGALPIEVDVSNLN
MGDVIDVYPYKGEVRNHETGELLATFELKTDVLIDEVRAGGRIPLIIGRGLT
TKAREALGLPHSDVFRQAKDVAESDRGFSLAQKMVGRACGVKGIRPGAYC
EPKMTSVGSQDTTGPMTRDELKDLACLGFSADLVMQSFCHTAAYPKPVDV
NTHHTLPDFIMNRGGVSLRPGDGVIHSWLNRMLLPDTVGTGGDSHTRFPIG
ISFPAGSGLVAFAAATGVMPLDMPESVLVRFKGKMQPGITLRDLVHAIPLY
AIKQGLLTVEKKGKKNIFSGRILEIEGLPDLKVEQAFELTDASAERSAAGCTI
KLNKEPIIEYLNSNIVLLKWMIAEGYGDRRTLERRIQGMEKWLANPELLEA
DADAEYAAVIDIDLADIKEPILCAPNDPDDARPLSAVQGEKIDEVFIGSCMT
NIGHFRAAGKLLDAHKGQLPTRLWVAPPTRMDAAQLTEEGYYSVFGKSGA
RIEIPGCSLCMGNQARVADGATVVSTSTRNFPNRLGTGANVFLASAELAAV
AALIGKLPTPEEYQTYVAQVDKTAVDTYRYLNFNQLSQYTEKADGVIFQT
AV

35 SEQIDNo. 128
MEFFKKTALAALVMGFSGAALALPNITILATGGTIAGGGDSATKSNYTVGK VGVENLVNAVPQLKDIANVKGEQVVNIGSQDMNDNVWLTLAKKINTDCD KTDGFVITHGTDTMEETAYFLDLTVKCDKPVVMVGAMRPSTSMSADGPFN LYNAVVTAADKASANRGVLVVMNDTVLDGRDVTKTNTTDVATFKSVNY GPLGYIHNGKIDYQRTPARKHTSDTPFDVSKLNELPKVGIVYNYANASDLP AKALVDAGYDGIVSAGVGNGNLYKSVFDTLATAAKTGTAVVRSSRVPTG ATTQDAEVDDAKYGFVASGTLNPQKARVLLQLALTQTKDPQQIQQIFNQY
SEQ ID No. 129
MKRAVITGLGIVSSIGNNQQEVLASLREGRSGITFSQELKDSGMRSHVWGN
VKLDTTGLIDRKVVRFMSDASIYAFLSMEQAIADAGLSPEAYQNNPRVGLI
AGSGGGSPRFQVFGADAMRGPRGLKAVGPYVVTKAMASGVSACLATPFKI
HGVNYSISSACATSAHCIGNAVEQIQLGKQDIVFAGGGEELCWEMACEFDA
MGALSTKYNDTPEKASRTYDAHRDGFVIAGGGGMVVVEELEHALARGAH
lYAEIVGYGATSDGADMVAPSGEGAVRCMKMAMHGVDTPIDYLNSHGTS
TPVGDVKELAAIREVFGDKSPAISATKAMTGHSLGAAGVQEAIYSLLMLEH
GFIAPSINIEELDEQAAGLNIVTETTDRELTTVMSNSFGFGGTNATLVMRKL
KD
SEQIDNo. 130
MKSVLKVSLAALTLAFAVSSHAADKKLVVATDTAFVPFEFKQGDKYVGFD
VDLWAAIAKELKLDYELKPMDFSGIIPALQTKNVDLALAGITITDERKKAID
FSDGYYKSGLLVMVKANNNDVKSVKDLDGKVVAVKSGTGSVDYAKANI
KTKDLRQFPNIDNAYMELGTNRADAVLHDTPNILYFIKTAGNGQFKAVGD
SLEAQQYGIAFPKGSDELRDKVNGALKTLRENGTYNEIYKKWFGTEPK
SEQIDNo. 131
MARKWLNLFAGAALSFAVAGNALADEGKITVFAAASLTNAMQDIATQFK
KEKGVDVVSSFASSSTLARQIEAGAPADLFISADQKWMDYAVDKKAIDTA
TRQTLLGNSLVVVAPKASVQKDFTIDSKTNWTSLLNGGRLAVGDPEHVPA
GIYAKEALQKLGAWDTLSPKLAPAEDVRGALALVERNEAPLGIVYGSDAV

36 ASKGVKVVATFPEDSHKKVEYPVAVVEGHNNATVKAFYDYLKGPQAAEIF KRYGFTIK
SEQIDNo. 132
MAIEIKVPDIGADEVEITEILVKVGDKVEAEQSLITVEGDKASMEVPSPQAGI
VKEIKVSVGDKTQTGALIMIFDSADGAADAAPAQAEEKKEAAPAAAPAAA
AAKDVNVPDIGSDEVEVTEILVKVGDKVEAEQSLITVEGDKASMEVPAPFA
GTVKEIKVNVGDKVSTGSLIMVFEVAGEAGAAAPAAKQEAAPAAAPAPAA
GVKEVNVPDIGGDEVEVTEVMVKVGDKVAAEQSLITVEGDKASMEVPAPF
AGVVKELKVNVGDKVKTGSLIMIFEVEGAAPAAAPAKQEAAAPAPAAKAE
APAAAPAAKAEGKSEFAENDAYVHATPLIRRLAREFGVNLAKVKGTGRKG
RILREDVQAYVKEAIKRAEAAPAATGGGIPGMLPWPKVDFSKFGEIEEVEL
GRIQKISGANLSRNWVMIPHVTHFDKTDITELEAFRKQQNEEAAKRKLDVK
ITPVVFIMKAVAAALEQMPRFNSSLSEDGQRLTLKKYINIGVAVDTPNGLV
VPVFKDVNKKGIIELSRELMTISKKARDGKLTAGEMQGGCFTISSIGGLGTT
HFAPIVNAPEVAILGVSKSAMEPVWNGKEFVPRLMLPISLSFDHRVIDGADG
ARFITIINNTLSDIRRLVM
SEQIDNo. 133
MKVKVLSLLVPALLVAGAANAAEVYNKDGNKLDLYGKVDGLHYFSDDK
SVDGDQTYMRLGFKGETQVTDQLTGYGQWEYQIQGNSAENENNSWTRVA
FAGLKFQDVGSFDYGRNYGVVYDVTSWTDVLPEFGGDTYGSDNFMQQRG
NGFATYRNTDFFGLVDGLNFAVQYQGKNGSVSGEGMTNNGREALRQNGD
GVGGSITYDYEGFGIGAAVSSSKRTDDQNSPLYIGNGDRAETYTGGLKYDA
NNIYLAAQYTQTYNATRVGSLGWANKAQNFEAVAQYQFDFGLRPSLAYL
QSKGKNLGVINGRNYDDEDILKYVDVGATYYFNKNMSTYVDYKINLLDD
NQFTRDAGINTDNIVALGLVYQF
SEQIDNo. 134
MSELNEKLATAWEGFTKGDWQNEVNVRDFIQKNYTPYEGDESFLAGATE
ATTTLWDKVMEGVKLENRTHAPVDFDTAVASTITSHDAGYINKQLEKIVG
LQTEAPLKRALIPFGGIKMIEGSCKAYNRELDPMIKKIFTEYRKTHNQGVFD
VYTPDILRCRKSGVLTGLPDAYGRGRIIGDYRRVALYGIDYLMKDKLAQFT

37 SLQADLENGVNLEQTIRLREEIAEQHRALGQMKEMAAKYGYDISGPATNA QEAIQWTYFGYLAAVKSQNGAAMSFGRTSTFLDVYIERDLKAGKITEQEA QEMVDHLVMKLRMVRFLRTPEYDELFSGDPIWATESIGGMGLDGRTLVTK NSFRFLNTLYTMGPSPEPNMTILWSEKLPLNFKKFAAKVSIDTSSLQYENDD LMRPDFNNDDYAIACCVSPMIVGKQMQFFGARANLAKTMLYAINGGVDE KLKMQVGPKSEPIKGDVLNYDEVMERMDHFMDWLAKQYITALNIIHYMH DKYSYEASLMALHDRDVIRTMACGIAGLSVAADSLSAIKYAKVKPIRDEDG LAIDFEIEGEYPQFGNNDPRVDDLAVDLVERFMKKIQKLHTYRDAIPTQSVL TITSNVVYGKKTGNTPDGRRAGAPFGPGANPMHGRDQKGAVASLTSVAKL PFAYAKDGISYTFSIVPNALGKDDEVRKTNLAGLMDGYFHHEASIEGGQHL NVNVMNREMLLDAMENPEKYPQLTIRVSGYAVRFNSLTKEQQQDVITRTF TQSM
SEQIDNo. 135
MSILIDKNTKVICQGFTGSQGTFHSEQAIAYGTKMVGGVTPGKGGTTHLGL
PVFNTVREAVAATGATASVIYVPAPFCKDSILEAIDAGIKLIITITEGIPTLDM
LTVKVKLDEAGVRMIGPNCPGVITPGECKIGIQPGHIHKPGKVGIVSRSGTL
TYEAVKQTTDYGFGQSTCVGIGGDPIPGSNFIDILEMFEKDPQTEAIVMIGEI
GGSAEEEAAAYIKEHVTKPVVGYIAGVTAPKGKRMGHAGAIIAGGKGTAD
EKFAALEAAGVKTVRSLADIGEALKTVLK
SEQIDNo. 176
MSSRKELANAIRALSMDAVQKAKSGHPGAPMGMADIAEVLWRDFLKHNP
QNPSWADRDRFVLSNGHGSMLIYSLLHLTGYDLPMEELKNFRQLHSKTPG
HPEVGYTAGVETTTGPLGQGIANAVGMAIAEKTLAAQFNRPGHDIVDHYT
YAFMGDGCMMEGISHEVCSLAGTLKLGKLIAFYDDNGISIDGHVEGWFTD
DTAMRFEAYGWHVIRDIDGHDAASIKRAVEEARAVTDKPSLLMCKTIIGFG
SPNKAGTHDSHGAPLGDAEIALTREQLGWKYAPFEIPSEIYAQWDAKEAGQ
AKESAWNEKFAAYAKAYPQEAAEFTRRMKGEMPSDFDAKAKEFIAKLQA
NPAKIASRKASQNAIEAFGPLLPEFLGGSADLAPSNLTLWSGSKAINEDAAG
NYIHYGVREFGMTAIANGISLHGGFLPYTSTFLMFVEYARNAVRMAALMK
QRQVMVYTHDSIGLGEDGPTHQPVEQVASLRVTPNMSTWRPCDQVESAV
AWKYGVERQDGPTALILSRQNLAQQERTEEQLANIARGGYVLKDCAGQPE

38 LIFIATGSEVELAVAAYEKLTAEGVKARVVSMPSTDAFDKQDAAYRESVLP KAVTARVAVEAGIADYWYKYVGLNGAIVGMTTFGESAPAELLFEEFGFTV DNVVAKAKELL
SEQIDNo. 136
MSKCSADETPVCCCMDVGTIMDNSDCTASYSRVFANRAEAEQTLAALTEK
ARSVESEPCKITPTFTEESDGVRLDIDFTFACEAEMLIFQLGLR
SEQIDNo. 137
MGLFNFVKDAGEKLWDAVTGQHDKDDQAKKVQEHLNKTGIPDADKVNI QIADGKATVTGDGLSQEAKEKILVAVGNISGIASVDDQVKTATPATASQFY TVKSGDTLSAISKQVYGNANLYNKIFEANKPMLKSPDKIYPGQVLRIPEE
said peptides being chosen, preferably, from the peptides having the sequence SEQ ID No.46 to 61 as defined hereinafter:
Peptide SEQ ID No. Amino acid sequence Location in SEQ ID No. 127
46 ILEIEGLPDLK 578-588
47 VADGATWSTSTR 779-791 Peptide SEQ ID No. Amino acid sequence Location in SEQ ID No. 128
48 TNTTDVATFK 185-194 Peptide SEQ ID No. Amino acid sequence Location in SEQ ID No. 129
49 LDTTGLIDR 54-62
50 VGLIAGSGGGSPR 99-111 Peptide SEQ ID No. Amino acid sequence Location in SEQ ID No. 130
51 AIDFSDGYYK 100-109 Peptide SEQ ID No. Amino acid sequence Location in SEQ ID No. 131
52 LGAWDTLSPK 160-169 Peptide SEQ ID No. Amino acid sequence Location in SEQ ID No. 132
53 FGEIEEVELGR 397-407 Peptide SEQ ED No. Amino acid sequence Location in SEQ ID No. 133
54 INLLDDNQFTR 339-349 Peptide SEQ ED No. Amino acid sequence Location in SEQ ED No. 134

39
55 LATAWEGFTK 8-17
Peptide SEQ ID No. Amino acid sequence Location in SEQ ED No. 135
56 DSILEAIDAGK 80-91
57 FAALEAAGVK 263-272 -
58 SLADIGEALK 276-285 Peptide SEQ ID No. Amino acid sequence Location in SEQ ID No. 176
59 TEEQLANIAR 529-538 Peptide SEQ ID No. Amino acid sequence Location in SEQ ID No. 136
60 AEAEQTLAALTEK 38-50
Peptide SEQ ID No. Amino acid sequence Location in SEQ ID No. 137
61 SGDTLSAISK 103-112
It should be noted, as previously indicated, that, for the typing, the method of the invention may also use at least one peptide having the sequence SEQ ID No.: 46 to 66 and 85 to 87, which are of use for determining the potential resistance to at least one antibiotic, the identification or the virulence, as previously indicated.
According to another preferred embodiment, the method of the invention allows
the characterization of Candida albicans.
In particular, the characterization of Candida albicans uses at least one peptide
as follows:
1. for the typing:
at least one peptide belonging to the alcohol dehydrogenase 1 protein (ADHl), the fructose-biphosphate aldolase protein (ALF), the lanosterol 14-alpha demethylase protein (CP51), F-box protein COSlll (CSlll), elongation factor 1-beta (EFIB), enolase 1 (ENOl), glyceraldehyde-3-phosphate dehydrogenase (G3P), heat shock protein SSAl (HSP71), heat shock protein SSBl (HSP75), pyruvate kinase (KPYK), lipase 8 (LIPS), muhiprotein-bridging factor 1 (MBFl), nuclear transport factor 2 (NTF2), phosphoglycerate kinase (PGK), peptidyl-prolyl cis-trans isomerase (PPIA), 60S ribosomal protein LI3 (RL13), 60S ribosomal protein L28 (RL28), 60S ribosomal protein L36 (RL36), 40S ribosomal protein S22 (RS22), triosephosphate isomerase (TPIS), having the following sequences SEQ ID No. 155 to 175, respectively:

40
SEQIDNo. 155
MSEQIPKTQKAVVFDTNGGQLVYKDYPVPTPKPNELLIHVKYSGVCHTDL
HARKGDWPLATKLPLVGGHEGAGVVVGMGENVKGWKIGDFAGIKWLNG
SCMSCEFCQQGAEPNCGEADLSGYTHDGSFEQYATADAVQAAKIPAGTDL
ANVAPILCAGVTVYKALKTADLAAGQWVAISGAGGGLGSLAVQYARAMG
LRVVAIDGGDEKGEFVKSLGAEAYVDFTKDKDIVEAVKKATDGGPHGAIN
VSVSEKAIDQSVEYVRPLGKVVLVGLPAHAKVTAPVFDAVVKSIEIKGSYV
GNRKDTAEAIDFFSRGLIKCPIKIVGLSDLPEVFKLMEEGKILGRYVLDTSK
SEQIDNo. 156 , MAPPAVLSKSGVIYGKDVKDLFDYAQEKGFAIPAINVTSSSTVVAALEAAR DNKAPIILQTSQGGAAYFAGKGVDNKDQAASIAGSIAAAHYIRAIAPTYGIP VVLHTDHCAKKLLPWFDGMLKADEEFFAKTGTPLFSSHMLDLSEETDDENI ATCAKYFERMAKMGQWLEMEIGITGGEEDGVNNEHVEKDALYTSPETVF AVYESLHKISPNFSIAAAFGNVHGVYKPGNVQLRPEILGDHQVYAKKQIGT DAKHPLYLVFHGGSGSTQEEFNTAIKNGVVKVNLDTDCQYAYLTGIRDYV TNKIEYLKAPVGNPEGADKPNKKYFDPRVWVREGEKTMSKRIAEALDIFHT KGQL
SEQIDNo. 157
MAIVETVIDGINYFLSLSVTQQISILLGVPFVYNLVWQYLYSLRKDRAPLVF
YWIPWFGSAASYGQQPYEFFESCRQKYGDVFSFMLLGKIMTVYLGPKGHE
FVFNAKLSDVSAEDAYKHLTTPVFGKGVIYDCPNSRLMEQKKFAKFALTT
DSFKRYVPKIREEILNYFVTDESFKLKEKTHGVANVMKTQPEITIFTASRSLF
GDEMRRIFDRSFAQLYSDLDKGFTPINFVFPNLPLPHYWRRDAAQKKISAT
YMKEIKSRRERGDIDPNRDLIDSLLIHSTYKDGVKMTDQEIANLLIGILMGG
QHTSASTSAWFLLHLGEKPHLQDVIYQEVVELLKEKGGDLNDLTYEDLQK
LPSVNNTIKETLRMHMPLHSIFRKVTNPLRIPETNYIVPKGHYVLVSPGYAH
TSERYFDNPEDFDPTRWDTAAAKANSVSFNSSDEVDYGFGKVSKGVSSPYL
PFGGGRHRCIGEQFAYVQLGTILTTFVYNLRWTIDGYKVPDPDYSSMVVLP
TEPAEIIWEKRETCMF

41 SEQIDNo. 158
MLKTDSLDFHSYLPPYRSLINPNARYDYRTHSLIPLTQNDLNLLRIAFQKKK EAPPSAFKMKYKSLLSDVSRTISMRLSNSNLLSSSSANNNNVLLSPPPSSSST LSTPCGNILNRAGTTSSNISKINNLSQNQTQNQLPLFPAELHIKNLPVEILDYI FYLVDDNLDYKSCMYTCKLFYFLAKPYYYENLVFTSTYRFAQFVTYLRVN SEVGQYVQSIDLSGIKPGYDEDEQEEGQEENAENGEEENGGGARDPQYLLG EIADNPHHERVDQFPRGKILAGWRDWKFKNNPLYTIHPSPSLTKIASNSQFS NVSSKSSRSTSSKSSSSTTKKFVKPFRYFKSRKRKMSYSGTTKLERKSPRLE QLQLDQYSSNWNKRVNSHPLINKFLLHYSTSKDLPIGYILHMINLCPNIVSL NLGNLSLSTDYEISRSTIHKYQNFDLINNYPKDLIYKVDNIMRLNDVDDVYS IDGSILRFGNINSGSSGSNWERNGSSSNNRILFKSNQSIASTASSVYSVTTFSK PIRKYNSLLPPLPQTVADISYLNKGDGKVYLSDLNLKEINSAYLKKINEDEIL SAIINVHGKRLIEYDTSLYQIPKPLNVDIAGTLKYINLSSMIWLNRKLIEKFLT RLLTKKSPELDMYGICYTDEFFDSDEQESDDDYEDSDDEEQRQCPIIYKQNL VIDFTDSGMYKSLPWAKRJDLNSFEGCQLANKIINNDLMTPQEQALRRERR RRGAIAANYLA
SEQIDNo. 159
MSFSDFSKVESIKSLNEFLADKSYIDGTTATQADVTVYKAFQKEFPQFTRWF
NHIASFTEEFEDLPAGKAPAASGSAAAAAEEEDDEDVDLFGSDDEVDEEAE
KLKQQRLAEYAAKKAAKGPKPAAKSIVTLDVKPWDDETDLDELLTNVKAI
EMEGLTWGAHQWIPVGFGIKKLQINLVVEDALVSLDDLQAAVEEDEDHVQ
STDIAAMQKL
SEQIDNo. 160
MSYATKIHARYVYDSRGNPTVEVDFTTDKGLFRSIVPSGASTGVHEALELR
DGDKSKWLGKGVLKAVANVNDIIAPALIKAKIDVVDQAKIDEFLLSLDGTP
NKSKLGANAILGVSLAAANAAAAAQGIPLYKHIANISNAKKGKFVLPVPFQ
NVLNGGSHAGGALAFQEFMIAPTGVSTFSEALRIGSEVYHNLKSLTKKKYG
QSAGNVGDEGGVAPDIKTPKEALDLIMDAIDKAGYKGKVGIAMDVASSEF
YKDGKYDLDFKNPESDPSKWLSGPQLADLYEQLISEYPIVSIEDPFAEDDW
DAWVHFFERVGDKIQIVGDDLTVTNPTRIKTAIEKKAANALLLKVNQIGTL

42 TESIQAANDSYAAGWGVMVSHRSGETEDTFIADLSVGLRSGQIKTGAPARS ERLAKLNQILRIEEELGSEAIYAGKDFQKASQL
SEQIDNo. 161
MAIKIGINGFGRIGRLVLRVALGRKDIEVVAVNDPFIAPDYAAYMFKYDST
HGRYKGEVTASGDDLVIDGHKIKVFQERDPANIPWGKSGVDYVIESTGVFT
KVEGAQKHIDAGAKKVIITAPSADAPMFVVGVNEDKYTPDLKIISNASCTT
NCLAPLAKVVNDTFGIEEGLMTTVHSITATQKTVDGPSHKDWRGGRTASG
NIIPSSTGAAKAVGKVIPELNGKLTGMSLRLPTTDVSVVDLTVRLKKAASYE
EIAPAIKKASEGPLKGVLGYTEDAVVSTDFLGSSYSSIFDEKAGILLSPTFVK
LISWYDNEYGYSTKVVDLLEHVA
SEQIDNo. 162
MSKAVGIDLGTTYSCVAHFANDRVEIIANDQGNRTTPSFVAFTDTERLIGDA
AKNQAAMNPANTVFDAKRLIGRKFDDPEVINDAKHFPFKVIDKAGKPVIQV
EYKGETKTFSPEEISSMVLTKMKEIAEGYLGSTVKDAVVTVPAYFNDSQRQ
ATKDAGTIAGLNVLRIINEPTAAAIAYGLDKKGSRGEHNVLIFDLGGGTFDV
SLLAIDEGIFEVKATAGDTHLGGEDFDNRLVNFFIQEFKRKNKKDISTNQRA
LRRLRTACERAKRTLSSSAQTSIEIDSLYEGIDFYTSITRARFEELCADLFRST
LDPVGKVLADAKIDKSQVEEIVLVGGSTRIPKIQKLVSDFFNGKELNKSINP
DEAVAYGAAVQAAILTGDTSSKTQDILLLDVAPLSLGIETAGGIMTKLIPRN
STIPTKKSETFSTYADNQPGVLIQVFEGERAKTKDNNLLGKFELSGIPPAPRG
VPQIEVTFDIDANGILNVSALEKGTGKTQKITITNDKGRLSKEEIDKMVSEAE
KFKEEDEKEAARVQAKNQLESYAYSLKNTINDGEMKDKIGADDKEKLTKA
IDETISWLDASQAASTEEYEDKRKELESVANPIISGAYGAAGGAPGGAGGFP
GAGGFPGGAPGAGGPGGATGGESSGPTVEEVD
SEQIDNo. 163
MADGVFQGAIGIDLGTTYSCVATYDSAVEIIANEQGNRVTPSFVAFTSEERL
IGDAAKNQAALNPKNTVFDAKRLIGRAFDDESVQKDIKSWPFKVVESNGQ
PLIEVEYLDETKTFSPQEISSMVLTKMKEIAEAKIGKKVEKAVVTVPAYFND
AQRQATKDAGAIAGLNVLRIINEPTAAAIAYGLGAGKSEKERHVLIFDLGG
GTFDVSLLNITGGVFTVKATAGDTHLGGQDFDTNLLEHFKKEFQKKTGNDI

43 S SD ARALRRLRTACERAKRSLS SGTQTTVEIDSLFDGEDFS ANITRARFEDIN SALFKSTLEPVEQVLEDAKISKSQVDEVVLVGGSTRIPKVQKLLSDFFDGKQ LEKSINPDEAVAYGAAVQGAILTGQSTNDDTKDLLLLDVIPLSLGVAMQGN VLAPVVPRNTTVPTIKRRTFTTVADHQTTVQFPVYQGERVNCSENTLLGEF DLKNIPPMQAGEPVLEAIFEVDANGILKVTAVEKSTGRSANITISNSIGRLST EEIEKMISDAEKFKSSDDAFAKRHEQKQKLEAYVASVESTVTDPVLSAKLK KSAKDKIEAALSDALQTLEIEESSADDYRKAELALKRAVTKGMATR
SEQIDNo. 164
FTIPPNHEMIFTTDDAYKTKCDDKVMIIDYKNITKVIAPGKIIYVDDGVLSFE
VISVDDQQTLKVRSLNAGMISSHKTANDVLELRVLSTSG
SEQIDNo. 165
MLFLLFLLITPIYAGLIFPTKPSSDPFYNPPKGFEKAAVGDILQSRETPKSITG
RFAPLKIQNSWQLLVRSEDSFGNPNAIVTTVIEPVNADPSKIASYQVFEDAA •
KADCAPSYALQFGSDLTTFVTQAEMYLMAPLLDQGYYVVSPDYEGPKSTF
TIGKQSGQAVLNSIRATLKSSKITNIKEDAKVVMWGYSGGSLASGWAAAL
QPSYAPELSSSLLGAALGGFVTNITATAQAADGTVFAGIVANALGGVANEY
PEFKSILQSDTDKKSVFDEFDSHCLADGVIDYINTSFLTGDNKIFKTGWDILK
SPTIAKIVEDNGLVYQKQLVPKIPIFVYHGSIDQIVPIVNVKKTYQNWCEGGI
SSLEFAEDGTNGHLTETVVGAPAALTWIIDRFNGKQTVSGCQHDKRLSNFQ
YPNISSSILKYFKVALDTMMSNGLGSDIQKDKITPDDLRKFLLGGW
SEQIDNo. 166
MSSDWDSVTIIGQKARVGGGGPRENVAKTSSQLNAARRAGLVVGTEKKY
GTANTKSNPEGQRLTKLDATDDVVAVKKVDVSVGKAIQQARQEKKLTQK
ELATKVNEKPNVINDYEAGRAIPNQQLLAKLERALGVKLRGKNIGEPLFAK
KK
SEQIDNo. 167
MSVDFNAVATEFCNFYYNQFDSDRSQLGNLYRNESMLTFETSQLQGARDI VEKLASLPFQKVAHRISTLDAQPASANGDILVMVTGELLIDEEQNAQRYSQ VFHLIPDNGSYYVFNDIFRLNYS

44
SEQIDNo. 168
MSLSNKLSVKDLDVAGKRVFIRVDFNVPLDGKTITNNQRIVAALPTIKYVE
EHKPKYIVLASHLGRPNGERNDKYSLAPVATELEKLLGQKVTFLNDCVGPE
VTKAVENAKDGEIFLLENLRYHIEEEGSSKDKDGKKVKADPEAVKKFRQEL
TSLADVYINDAFGTAHRAHSSMVGLEVPQRAAGFLMSKELEYFAKALENP
ERPFLAILGGAKVSDKIQLIDNLLDKVDMLIVGGGMAFTFKKILNKMPIGDS
LFDEAGAKNVEHLVEKAKKNNVELILPVDFVTADKFDKDAKTSSATDAEG
IPDNWMGLDCGPKSVELFQQAVAKAKTIVWNGPPGVFEFEKFANGTKSLL
DAAVKSAENGNIVIIGGGDTATVAKKYGVVEKLSHVSTGGGASLELLEGK
DLPGVVALSNKN
SEQIDNo. 169
MSTVYFDVSADGQKLGKITFKLYDDVVPKTAENFRALCTGEKGFGYKGSIF
HRVIPQFMLQGGDFTNFNGTGGKSIYGTKFADENFVKRHDRPGLLSMANA
GPNTNGSQFFITTVPCPWLDGKHVVFGEVTDGLDIVKKIESFGSGSGATSKK
IVIEESGQL
SEQIDNo. 170
MAISKNLPLLNNHFRKHWQERVRVHFDQAGKKASRRQSRLRKAAKIAPRP
IDALRPVVRAPTVKYNRKVRAGRGFTLAELKAVGIAPKYARTIGISVDHRR
QNKSQETFDANVARLQEYKSKLVIFDKKTKASEVASFEQVDVSATFPVEQP
APESGLRAVEVPEQTAYRTLRLARNEKKYKGIREKRAKEKAEAEAEKAKK
SEQIDNo. 171 EKKDEYLSKSSASAAPVIDTLAHGYGKVLGKGRLPEVPVIVKARFVSKLAE
EKSESLVVLSN
SEQIDNo. 172
MAKSGIAAGVNKGRKTTAKEVAPKISYRKGASSQRTVFVRSIVKEVAGLAP
YERRLIELIRNAGEKRAKKLAKKRLGTHKRALRKVEEMTQVIAESRRH

45
SEQIDNo. 173
MKYLAAYLLLVQGGNTSPSASDITALLESVGVEAEESRLQALLKDLEGKDL QELIAEGNTKLASVPSGGAAAGGASASTGAAAGGAAEAEEEKEEEAKEES DDDMGFGLFD
SEQIDNo. 174
MTRTSVLADALNAINNAEKTGKRQVLIRPSSKVIIKFLTVMQKHGYIGEFEY IDDHRSGKIVVQLNGRLNKCGVIQPRFNVKINDIERWTDNLLPARQFGYVIL TTSAGIMDHEEARRKHVSGKILGFVY
SEQIDNo. 175
MARQFFVGGNFKANGTKQQITSIIDNLNKADLPKDVEVVICPPALYLGLAV
EQNKQPTVAIGAQNVFDKSCGAFTGETCASQILDVGASWTLTGHSERRTIIK
ESDEFIAEKTKFALDTGVKVILCIGETLEERKGGVTLDVCARQLDAVSKIVS
DWSNIVVAYEPVWAIGTGLAATPEDAEETHKGIRAHLAKSIGAEQAEKTRI
LYGGSVNGKNAKDFKDKANVDGFLVGGASLKPEFVDIIKSRL
said peptides being chosen from the peptides having the sequence SEQ ID No.88 to 125 as defined hereinafter:
Peptide SEQ ID No. Amino acid sequence Location in SEQ ID No. 156
88 ADEEFFAK 125-132
97 lAEALDIFHTK 346-356
Peptide SEQ ID No. Amino acid sequence Location in SEQ ID No. 163
89 AFDDESVQK 79-87 Peptide SEQ ID No. Amino acid sequence Location in SEQ ID No. 160
90 AKID\A/DQAK 81-90
98 IDWDQAK 83-90
99 lEEELGSEAIYAGKDFQK 419-436
112 VGDKIQIVGDDLTVTNPTR 315-333
Peptide SEQ ID No. Amino acid sequence Location in SEQ ID No. 170
91 AVEVPEQTAYR 160-170
107 SQETFDANVAR 105-115

46
Peptide SEQ ED No. Amino acid sequence Location in SEQ ID No. 174
92 CGVIQPR 72-78
114 WTDNLLPAR 89-97
Peptide SEQ ID No. Amino acid sequence Location in SEQ ID No. 173
93 DLQELIAEGNTK 50-61
Peptide SEQ ID No. Amino acid sequence Location in SEQ ID No. 169
94 FADENFVKR 81-89
100 lESFGSGSGATSK 140-152
Peptide SEQ ID No. Amino acid sequence Location in SEQ ID No. 175
95 FALDTGVK 115-122
Peptide SEQ ID No. Amino acid sequence Location in SEQ ID No. 171
^^ GRLPEVPVIVK 32-42
Peptide SEQ ID No. Amino acid sequence Location in SEQ ED No. 167
101 LASLPFQK 54-61
Peptide SEQ ID No. Amino acid sequence Location in SEQ ED No. 166
102 LDATDDWAVK 66-76
Peptide SEQ ID No. Amino acid sequence Location in SEQ ID No. 168
103 NVEHLVEK 264-271
110 SVELFQQAVAK 319-329
Peptide SEQ ED No. Amino acid sequence Location in SEQ ID No. 172
104 SGIAAGVNK 4-12
Peptide SEQ ED No. Amino acid sequence Location in SEQ ED No.161
105 SGVDYVIESTGVFTK 89-103
115 YKGEVTASGDDLVIDGHK 55-72
Peptide SEQ ED No. Amino acid sequence Location in SEQ ED No. 158
108 SSSSTTKK 326-333
Peptide SEQ ID No. Amino acid sequence Location in SEQ ED No. 159
106 SLNEFLADK 14-22
Peptide SEQ ED No. Amino acid sequence Location in SEQ ED No. 162
109 STLDPVGK 311-318
Peptide SEQ ID No. Amino acid sequence Location in SEQ ED No. 164
111 TANDVLELR 78-86
Peptide SEQ ID No. Amino acid sequence Location in SEQ ED No. 155
113 WAIDGGDEK 200-209
I I ; \

47
116 YVLDTSK 344-350
Peptide SEQ ED No. Amino acid sequence Location in SEQ ID No. 157
117 AVIYDCPNSR 399-414
177 GHYVLVFPGYAHTSER 399-414
118 GHYVLVSPGYAHTSER 399-414
119 GVIYDCPNSR 129-138
120 GVSSPYLPFGGGR 455-467
121 GVSSPYLPFSGGK 455-467
122 GVSSPYLPFSGGR 455-467 Peptide SEQ ID No. Amino acid sequence Location in SEQ ID No. 165
123 AAVGDILQSR 37-46
124 JTPDDLR 447-453
125 TGWDILK 304-310
2. For the potential resistance to at least one antibiotic
at least one peptide belonging to the lanosterol 14-alpha demethylase protein (CP51), having the sequence SEQ ID No. 157, said peptides being chosen, preferably, from the peptides having the sequences SEQ ID No. 117 to 122 and 177, as defined above.
3. For the virulence:
at least one peptide belonging to the lipase 8 protein (LIPS) having the sequence SEQ ID No. 163, said peptides being chosen, preferably, from the peptides having the sequence SEQ ID No.123 to 125, as defined above.
4. For the identification:
at least one peptide belonging to the alcohol dehydrogenase 1 protein (ADHl), the fructose-biphosphate aldolase protein (ALF), F-box protein COSlll (CSlll), elongation factor 1-beta (EFIB), enolase 1 (ENOl), glyceraldehyde-3-phosphate dehydrogenase (G3P), heat shock protein SSAl (HSP71), heat shock protein SSBl (HSP75), pyruvate kinase (KPYK), multiprotein-bridging factor 1 (MBFl), nuclear transport factor 2 (NTF2), phosphoglycerate kinase (PGK), peptidyl-prolyl cis-trans isomerase (PPIA), 60S ribosomal protein L13 (RL13), 60S ribosomal protein L28

48 (RL28), 60S ribosomal protein L36 (RL36), 40S ribosomal protein S22 (RS22), triosephosphate isomerase (TPIS), having the sequences SEQ ID No. 155, 156, 158 to 164, and 166 to 175, said peptides being chosen, preferably, from the peptides having the sequence SEQ ID No.88 to 116, as defined above.
The peptides that are of use for the purposes of the invention, having the sequence SEQ ID No.2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 19, 20, 21, 23, 24, 26, 27, 29, 30, 32, 34, 35, 37, 38, 40, 42, 44 to 125 and 177 are novel and constitute another subject of the invention.
The method of the invention and its advantages will emerge from the rest of the present description relating various nonlimiting examples of implementation of the method of the invention.
Example 1: Identification of microorganisms from a sample by means of biochemical profile
1. Culturing the sample on a culture medium
The optimum culture media and the optimum cultiure conditions are different according to the species of microorganism. By default, the sample is inoculated onto various media:
o Columbia agar with sheep blood (bioMerieux ref 43041) for 18 to 24 h
at 35°C, with or without anaerobic conditions; o TSA agar (bioMerieux reference 43011) for 18 to 24 h at 37°C.
2. Identification of microorganisms
The identification is implemented as follows:
1. Selection of isolated colonies.
2. While observing aseptic conditions, transfer of 3.0 ml of aqueous sterile saline solution (containing 0.45-0.50% of NaCl, at pH 4.5 to 7.0) into a transparent plastic (polystyrene) test tube.
3. Using a sterile cotton bud or a sterile swab, transfer of a sufficient number of identical colonies into the tube of saline solution prepared in step 2 and

49 adjustment of the bacterial suspension to between 0.50 and 0.63 McFarland with a calibrated VITEK 2 DENSICHEK.
4. Positioning of the tube of bacterial suspension and of a VITEK 2 identification card on a VITEK 2 cassette.
5. Loading of the cassette into the VITEK 2 instrument.
6. The filling, sealing, incubation and reading operations are automatic.
7. Acquisition of a biochemical profile.
8. Identification with the VITEK 2 system, carried out by comparison with biochemical profiles of known strains.
Example 2; Identification of microorganisms from a sample by means of MALDI-TOF
The identification is implemented as follows:
1. Transfer, using a 1 |al loop, of a portion of microorganism colony obtained according to example 1, and uniform deposition on a plate for mass spectrometry by MALDI-TOF.
2. Covering of the deposit with 1 f^l of matrix. The matrix used is a saturated solution of HCCA (alpha-cyano-4-hydroxycinnamic acid) in an organic solvent (50% acetonitrile and 2.5% trifluoroacetic acid).
3. Drying at ambient temperature.
4. Introduction of the plate into the mass spectrometer.
5. Acquisition of a mass spectrum.
6. Comparison of the spectrum obtained with the spectra contained in a knowledge base.
7. Identification of the microorganism by comparison of the peaks obtained with those of the knowledge base.
Example 3; Identification of microorganisms from a sample by means of ESI-MS
The identification is implemented as follows:
1. Sampling of a microorganism colony, obtained according to example 1, and suspension in 100 \il of demineralized water.
2. Centrifugation at 3000g for 5 minutes.

50
3. Removal of the supernatant.
4. Resuspension in 100 \il of demineralized water.
5. Centrifugation at 3000g for 5 minutes.
6. Removal of the supernatant.
7. Resuspension in 100 |il of a mixture of acetonitrile, demineralized water and formic acid (50/50/0.1%).
8. Filtration with a filter having a pore size of 0.45 |j,m.
9. Injection into a mass spectrometer in single MS mode.
10. Acquisition of a mass spectrum.
11. Comparison of the spectrum obtained with the spectra contained in a knowledge base.
12. Identification of the microorganism by reference to reference spectra.
Example 4; Obtaining of digested proteins from microorganisms
Conventionally, the following protocol is implemented in 11 steps:
1. Sampling of a microorganism colony, obtained according to example 1, and suspension in 10 to 100 [il of a solution of 6M guanidine hydrochloride, 50 mM Tris-HCl, pH=8.0.
2. Addition of dithiothreitol (DTT) in order to obtain a final concentration of 5mM.
3. Reduction for 20 minutes at 95 °C in a water bath.
4. Cooling of the tubes to ambient temperature.
5. Addition of iodoacetamide in order to obtain a final concentration of 12.5 mM.
6. Alkylation for 40 minutes at ambient temperature and in the dark.
7. Dilution by a factor of 6 with a 50 mM, NH4HCO3 solution, pH=8.0 in order to obtain a final guanidine hydrochloride concentration of IM.
8. Additionofl |ag of trypsin.
9. Digestion at 37°C for 6 hours overnight.
10. Addition of formic acid to a pH of less than 4 in order to stop the reaction.
11. Ultracentrifugation at 100 OOOg for 30 minutes.

51 Example 5: Characterization of 5. aureus samples;
After having established the one or more species of the samples according to any one of the methods described in examples 1 to 3, the species listed below are analyzed.
Thirteen strains of S. aureus are analyzed in order to confirm their identification and to establish their characteristics:
ID2 ID3
ID4 ID2a
lD3a ID4a
AST7 AST8
AST13 AST14
VIR5 VIR6
VIR7
The same method of analysis is applied to species that do not belong to the S. aureus species, in order to serve as a negative control:
• E. coli
ID7 AST2
• S. pneumoniae VIR21
• C. difficile VIR26
Each sample is treated according to example 4, then a volume of 5 |il of digested proteins is injected and analyzed according to the following conditions:
• Agilent 1100 series chromatographic system from the company Agilent Technologies (Massy, France).
• Waters Symmetry CI8 column, 2.1 mm internal diameter, 100 mm long, particle size 3.5 |j.m.
• Solvent A: H2O + 0.1 % formic acid.
• Solvent B: ACN + 0.1% formic acid.
• HPLC gradient defined in TABLE 1 hereinafter:

52
TABLE 1
Time I Flow rate (|il) I Solvent A (%) I Solvent B (%)
0 300 95 5
25 300 60 40
27 300 0 100
35 300 0 Too
35l 300 95 5
45 300 95 5
• The eluate at the output of the chromatographic column is directly injected into the ionization source of the QTRAP® 5500 mass spectrometer from the company Applied Biosystems (Foster City, United States of America).
• The peptides, resulting from the digestion of the proteins of the microorganism, are analyzed by mass spectrometry in MRM mode. Only the peptides indicated in TABLE 2 are detected. For this, the first-generation fragment(s) indicated in TABLE 2 is (are) detected. The application, to which each transition, i.e. each peptide associated with its first-generation fragment, makes it possible to respond, is specified in the clinical interest column of TABLE 2 with the letters I, T, R and V. I denotes the confirmation of the identification of the microorganism, T the typing, R the resistance to at least one antibiotic and V the detection of virulence factors.
TABLE 2:
Transition „ . ■ T> ^-j First-generation Clinical
, Protein Peptide j- ^ •
number fragment ion interest
1 protein A DDPSQSANVLGEAQK y9 singly charged T ~
2 protein A DDPSQSANVLGEAQK ylS singly charged T
3 protein A DQQSAFYEILNMPNLNEEQR y8 singly charged T
4 protein A DQQSAFYEILNMPNLNEEQR y8 doubly charged T
5 protein A DDPSQSTNVLGEAK y9 singly charged T
6 protein A DDPSQSTNVLGEAK yl2 singly charged T
7 protein A EQQNAFYEILNMPNLNEEQR y8 singly charged T
8 protein A EQQNAFYEn.NMPNLNEEQR yS doubly charged T
9 protein A DDPSQSANLLAEAK y9 singly charged T
10 protein A DDPSQSANLLAEAK yl2 singly charged T
11 protein A DDPSVSK y5 singly charged T
~ 12 I protein A | DDPSVSK | y4 singly charged | T ~

53
13 protein A lAADNK y4 singly charged T
14 protein A LAADNK y5 singly charged T
15 PBP2a lYNSLGVK y6 singly charged R + T
16 PBP2a lYNSLGVK y7 singly charged R + T
17 PBP2a DINIQDR y5 singly charged R + T
18 PBP2a DINIQDR y4 singly charged R + T
19 PBP2a ELSISEDYIK y6 singly charged R + T
20 PBP2a ELSISEDYIK y8 singly charged R + T
21 PBP2a FQITTSPGSTQK y9 singly charged R + T
22 PBP2a FQITTSPGSTQK y6 singly charged R + T
23 PBP2a ILTAMIGLNNK yS singly charged R + T
24 PBP2a ILTAMIGLNNK y9 singly charged R + T
25 PBP2a YEVVNGNIDLK yS singly charged R + T
26 PBP2a YEVVNGNIDLK y9 singly charged R + T
27 PBP2a VALELGSK y6 singly charged R + T
28 PBP2a VALELGSK y7 singly charged R + T
29 PBP2a SYANLIGK y6 singly charged R + T
30 LukS TNDPNVDLINYLPK y8 singly charged V + T
31 LukS TNDPNVDLINYLPK yll singly charged V + T
32 LukS SVQWGIK y5 singly charged V + T
33 LukS SVQWGIK y4 singly charged V + T
34 LukS ANSFITSLGK y8 singly charged V + T
35 LukS ANSFITSLGK y6 singly charged V + T
36 LukF MPVLSR y5 singly charged V + T
37 LukF MPVLSR y3 singly charged V + T
38 LukF GNFNPEFIGVLSR y8 singly charged V + T
39 LukF GNFNPEFIGVLSR y9 singly charged V + T
40 RL30 LQITLTR y5 singly charged I + T
41 RL30 LQITLTR y6 singly charged I + T
42 RL30 TNSSVWEDNPAIR y8 singly charged I + T
43 RL30 TNSSVWEDNPAIR y8 singly charged I + T
44 RL331 VNVTLAC[CAM]TEC[CAM]GDR y8 singly charged I + T
45 RL331 NYITTK y5 singly charged I + T
46 RL331 NYITTK y4 singly charged I + T
47 SSAA2 AGYTVNNTPK y6 singly charged I + T
48 SSAA2 AGYTVNNTPK y7 singly charged I + T
49 SSAA2 AGYTVNNTPK y8 singly charged I + T
50 SSAA2 AGYTVNNTPK y9 singly charged I + T
51 Y772 ATDFIDKVK y6 singly charged I + T
52 Y772 ATDFIDKVK y8 singly charged I + T
53 ATL AYLAVPAAPK y7 singly charged I + T
54 ATL AYLAVPAAPK y8 singly charged I + T
55 EFTU TVGSGWTEIDC y7 singly charged 1 + T
56 EFTU TVGSGWTEIDC yS singly charged I + T
57 EFTU TVGSGWTEIIK y9 singly charged I + T
58 Y772 EFVENAKEK y6 singly charged I + T
59 Y772 EFVENAKEK y7 singly charged I + T
60 Y772 EFVENAKEK y8 singly charged I + T
61 ISAA LSNGNTAGATGSSAAQIMAQR y7 singly charged I + T
~ 62 I ISAA ILSNGNTAGATGSSAAQIMAQRI yS singly charged | 1 + T

54
63 I ATL I LYSVPWGTYK I y6 singly charged I I + T
64 ATL LYSVPWGTYK y7 singly charged I + T
65 ATL LYSVPWGTYK y8 singly charged I + T
66 Y197A NITQDQDIHAVPK y8 singly charged I + T
67 Y197A NITQDQDIHAVPK y7 singly charged I + T
68 Y197A NITQDQDIHAVPK y6 singly charged I + T
69 Y197A LDSKDVSR y6 singly charged I + T
70 ~^197A I LDSKDVSR | y7 singly charged | I + T
The charge state of the precursor peptide, its retention time and the transitions, i.e. the
ratios (ni/z)i in Ql and (m/z)2 in Q3, are indicated in TABLE 3. The colHsion energy
used to fragment the precursor ion is also indicated in TABLE 3.
TABLE 3:
Transition Precursor charge Retention (m/z) filtered (m/z) filtered Collision
number state time inQl in Q3 energy
1 ' 2 8.2 ~ 779.87 929.51 " 39
2 2 82 779.87 664.84 39
3 3 17.9 813.71 999.49 45
4 3 17.9 813.71 500.24 45
5 2 7^9 730.85 918.49 37
6 2 7^9 730.85 615.82 37
7 3 16.5 827.39 999.49 45
8 3 16.5 827.39 500.25 34
9 2 10 729.86 916.51 37
10 2 10 729.86 614.83 37
11 2 n 374.18 517.3 21
12 2 H 374.18 420.25 21
13 2 8.9 316.17 447.22 19
14 2 8^9 316.17 518.26 19
15 2 6.7 447.26 617.36 25
16 2 6^ 447.26 780.43 25
17 2 6,2 437.22 645.33 24
18 2 62 437.22 531.3 24
19 2 11.1 598.81 754.36 31
20 2 11.1 598.81 954.48 31
21 2 7.4 647.84 906.45 34
22 2 7;4 647.84 617.3 26
23 2 11.8 594.34 860.47 31
24 2 11.8 594.34 961.51 31
25 2 10.1 632.33 872.48 33
26 2 10.1 632.33 971.55 33
27 2 X4 408.74 646.38 23
28 2 lA 408.74 717.41 23
29 2 8,2 433.24 615.38 24
30 2 14.9 808.42 975.55 41
31 2 14.9 808.42 643.4 41
32 I 2 I 8.2 I 409.23 | 631.36 | 23

55
33 I 2 I 8.2 I 409.23 I 503.3 I 23
34 2 10.3 519.28 852.48 28
35 2 10.3 519.28 618.4 28
36 2 6A 351.7 571.36 20
37 2 6,4 351.7 375.24 20
38 2 14.3 725.38 920.52 37
39 2 14.3 725.38 509.29 37
40 2 92 422.77 603.38 24
41 2 9.2 422.77 731.44 24
42 2 8 750.89 913.47 38
43 3 8 500.93 913.47 29
44 2 7,9 747.84 968.36 38
45 2 2,8 370.2 625.36 21
46 2 2,8 370.2 462.29 21
47 2 3,9 532.77 672.37 28
48 2 3.9 532.77 773.42 28.442
49 2 3,9 532.77 936.48 28.442
50 2 3,9 532.77 993.5 28.442
51 2 7.2 518.79 749.46 28
52 2 12 518.79 965.53 27.827
53 2 9 500.79 653.4 27
54 2 9 500.79 766.48 27.035
55 2 11.2 601.85 801.51 31
56 2 11.2 601.85 858.53 31.482
57 2 11.2 601.85 945.56 31.482
58 2 23 547.28 718.37 29
59 2 23 547.28 817.44 29.08
60 2 23 547.28 964.51 29.08
61 3 8.7 669.33 817.43 37
62 3 8J 669.33 888.47 37.466
63 2 12.2 607.32 751.38 32
64 2 12.2 607.32 850.45 31.722
65 2 12.2 607.32 937.48 31.722
66 2 63 739.88 907.5 38
67 2 63 739.88 779.44 37.555
68 2 6,3 739.88 664.41 37.555
69 2 2 460.25 691.37 25.251
70 I 2 I 2 I 460.25 | 806.4 | 25.251
• The other machine parameters used are the following:
Scan type: MRM
Polarity: Positive
Ionization source: Turbo V;^'^ (Applied BioSystems)
Ql setting: Filtering with unit resolution
Q3 setting: Filtering with unit resolution
Inter-scan pause: 5.00 msec

56
Scan speed: lODa/s
Curtain gas: 50.00 psi
Cone voltage: 5500.00 V
Source temperature: 500.00°C
Nebulizing gas: 5 0.00 psi
Heating gas: 40.00 psi
Dynamic filling: activated
Declustering potential (DP): 100.00 V
Entry potential before QO (EP): 6.00 V
Collision cell exit potential (CXP): 11 V
Total cycle time: 1.6 sec
The areas obtained for each of the transitions and for each of the microorganisms studied were measured. All the transitions with an area greater than or equal to 1000 (arbitrary units) are considered to be positive and have been denoted "1" in tables 4A and 4B. All the transitions with an area less than 1000 are considered to be negative and have been denoted 0 in tables 4A and 4B. When no signal peak was observed, the transition was noted as negative.
The positive-transition number is then summed for the applications I, R and V and reported in TABLE 5:
TABLE 4A:
ID2 ID3 ID4 ID2a IDS a ID4a AST7 AST8
Transition S. aureus S. aureus S. aureus S. aureus S. aureus S. aureus S. aureus S. aureus
1 0 1 1 0 1 1 0 ~ 0
2 0 1 1 0 1 1 0 0
3 0 1 1 0 1 1 0 0
4 0 1 1 0 1 1 0 0
5 1 1 1 1 1 1 0 0
6 1 1 1 1 1 1 0 1
7 1 1 1 1 1 1 0 0
8 1 1 1 1 1 1 0 0
9 1 1 1 1 1 1 0 0
10 1 I 1 1 1 1 0 0
11 0 0 0 0 0 0 1 0
~12|o|o|o|o|o|o|o|o~

57
13 I 1 I 1 I 1 I 0 I 1 I 1 I 0 I 0
14 1 1 1 1 I 1 I 0
15 1 0 1 1 0 1 0 0
16 0 0 1 0 0 1 0 0
17 1 0 1 1 0 1 0 0
18 1 0 1 1 0 1 0 0
19 1 0 1 1 0 1 0 0
20 1 0 1 1 0 1 0 0
21 1 0 1 1 0 1 0 0
22 1 0 1 1 0 1 0 0
23 1 0 1 1 0 1 0 0
24 1 0 1 1 0 1 0 0
25 1 0 1 1 0 1 0 0
26 1 0 1 1 0 1 0 0
27 1 1 1 1 1 1 0 0
28 1 0 1 1 0 1 0 0
29 1 0 1 1 0 1 0 0
30 0 0 0 0 0 0 0 0
31 0 0 0 0 0 0 0 0
32 0 0 0 0 0 0 0 0
33 0 0 1 0 0 0 0 0
34 0 0 0 0 0 0 0 0
35 0 0 0 0 0 0 0 1
36 0 0 0 0 0 0 1 1
37 0 0 0 0 0 1 0 0
38 0 0 0 0 0 1 0 0
39 0 0 0 0 0 0 0 0
40 1 1 1 1 1 1 1 1
41 1 1 1 1 1 1 1 1
42 1 1 1 1 1 1 1 1
43 1 1 1 1 1 1 1 1
44 1 1 1 1 1 1 1 1
45 1 1 1 1 1 1 1 1
46 \ 1 1 1 I 1 I 1
47 1 1 1 I 1 1 \ 1
48 1 1 1 1 1 1 \ 1
49 1 1 1 1 1 1 1 1
50 1 I 1 1 1 1 1 1
51 1 I 1 1 1 1 1 1
52 1 1 1 1 1 1 1 1
53 1 1 1 1 1 1 1 1
54 1 1 1 1 1 1 1 1
55 1 1 1 1 1 1 1 1
56 1 1 1 1 1 1 1 1
57 1 1 1 1 1 1 1 1
58 1 1 1 1 1 1 1^ 1
59 1 1 1 1 1 1 1 1
60 1 1 1 1 1 1 1 1
61 1 \ 1 1 1 1 1 1
62 I 1 I 1 I 1 I 1 I 1 I 1 I 1 I 1

58
63 I 1 I 1 I 1 I 1 I 1 I 1 I 1. I 1
64 1 1 \ 1 1 1 0 0
65 1 1 1 1 1 I 1 1
66 1 1 1 1 1 1 1 1
67 I 1 1 1 1 1 1 1
68 1 1 1 1 1 1 1 1
69 1 1 1 1 1 1 1 1
70 I 1 I 1 I 1 I 1 I 1 I 1 I 1 I 1
TABLE 4B:
AST13 AST14 VIR5 VIR6 VIR7 ID? AST2 VIR21 VIR26
_ ^
Transition 5. aureus S. aureus S. aureus S. aureus S. aureus E. coUE. coli . ,.^/.' .,
pneumoniae difficile
1 0 0 0 0 OOP 0 0
2 0 0 0 1 OOP P P
3 P P P P P P P P P
4 P P P P P P P P P
5 P P P 1 P P P P P
6 1 P P 1 P P P P P
7 P P P 1 P P P P P
8 P P P 1 P P P P P
9 P P P 1 P P P P P
IP P P P 1 P P P P P
11 P 1 1 1 P P P P P
12 P 1 1 P P P P P P
13 P P P P P P P P P
14 1 P P 1 P P P P P
15 1 P P P P P P P P
16 1 P P P P P P P P
17 1 1 P P P P P P P
18 1 P P P P P 1 P P
19 1 1 P P P P P P P
2P 1 1 P P P P P P P
21 1 1 P P P P P P P
22 1 P P P P P P P P
23 1 P P P P P 1 P P
24 1 1 P 1 P P P P p
25 1 1 P P P P P P P
26 1 P P P P P P P p
27 1 1 P P P P P P 1
28 1 1 P P P P P P p
29 1 1 P P P P P P P
3P P P 1 P 1 P P P p
^31 |p|p|l|p|l|p|p| P |p

59
32|0|0|l|0|l|0|0| 0 |0
33 0 0 1 0 1 0 0 0 0
34 0 0 1 0 10 0 0 0
35 0 0 1 0 1 0 0 0 0
36 1 1 1 1 1 0 0 0 0
37 0 0 1 0 1 0 0 0 0
38 0 0 1 0 1 0 0 0 0
39 0 0 1 0 1 0 0 0 0
40 1 1 1 1 1 0 0 0 0
41 1 1 1 1 1 0 0 1 0
42 1 1 1 1 1 0 0 0 0
43 0 1 0 1 0 0 0 0 0
44 1 1 1 1 1 0 0 0 0
45 1 1 0 1 1 0 0 0 0
46 1 1 1 1 1 0 0 0 0
47 1 1 1 1 1 0 0 0 0
48 1 1 0 1 10 0 0 0
49 1 1 0 1 0 0 0 0 0
50 1 0 0 1 0 0 0 0 0
51 1 1 0 1 10 0 0 0
52 1 1 0 1 110 0 0
53 I 1 1 1 10 0 0 0
54 1__^ 1 1 1 0 0 0 0
55 1 1 L____J 10 0 0 0
56 1 1 1 1 1 0 0 0 0
57 1 1 1 1 10 0 0 0
58 1 1 0 1 1 0 0 0 0
59 1 1 0 1 1 0 0 0 0
60 1 1 0 1 1 0 0 0 0
61 1 1 0 1 1 0 0 0 0
62 1 1 0 1 1 0 0 0 0
63 1 1 1 1 1 0 0 0 0
64 1 0 0 0 0 0 0 0 0
65 1 1 1 1 1 0 0 0 0
66 1 1 0 1 1 0 0 0 0
67 0 1 0 1 0 0 0 0 0
68 1 1 0 1 0 0 0 0 0
69 1 1 1 1 1 0 0 0 0
70 |l|l|o|l|l|o|o| 0 [0

60
TABLE 5:
I I I R V
ID2 I S. aureus " 30 14 0
ID3 S. aureus 30 1 0
ID4 S. aureus 30 15 1
ID2a S. aureus 30 14 0
ID3a S. aureus 30 1 0
ID4a S. aureus 30 IJ 2
AST7 S. aureus 29 0 1
AST8 S. aureus 29 0 2
AST 13 S. aureus 28 15 1
AST 14 S. aureus 28 9 1
VIR5 S. aureus 13 0 10
VIR6 S. aureus 29 1 1
V1R7 S. aureus 24 0 10
ID7 E. coli 1 0 0
AST2 E. coli 0 2 0
VIR21 S. pneumoniae 1 0 0
VIR26 I C. difficile | 0 | 1 0
All the S. aureus samples exhibit more than 12 positive transitions in the I category. All these samples are therefore confirmed as indeed belonging to the S. aureus species.
On the other hand, the samples ID7, AST2, VIR21 and VIR26 exhibit less than two positive transitions in the I category, these samples are therefore confirmed as not belonging to the S. aureus species.
The ID2, ID4, ID2a, ID4a, AST 13 and AST14 strains of 5'. aureus exhibit more than eight positive transitions for the R category, they therefore express the penicillin binding protein (PBP2a), this being synonymous with a mechanism of resistance to group M penicillins (e.g. methicillin).
On the other hand, the IDS, ID3a, AST7, AST8, VIR5, VIR6 and VIR7 strains of S. aureus exhibit less than three positive transitions for the V category, they therefore do not express PBP2a. These strains are therefore sensitive to an antibiotic such as a group M penicillin.
The ID7, AST2, VIR21 and VIR26 strains which do not belong to the S. aureus species also exhibit less than three transitions for the R category, they do not therefore express PBP2a, thereby confirming the specificity of the method.

61
The VIR5 and VIR7 samples of S. aureus exhibit more than nine positive transitions in the V category, they therefore express the Panton-Valentine-Leukocidin (PVL) protein.
On the other hand, the ID2, ID3, ID4, ID2a, ID3a, ID4a, AST7, AST8, AST13, AST 14 and VIR6 strains of S. aureus exhibit less than three positive transitions, they do not therefore express PVL. These strains do not therefore have the virulence properties related to PVL.
The ID7, AST2, VIR21 and VIR26 strains which do not belong to the S. aureus species also exhibit less than three transitions for the V category, they do not therefore express PVL, thereby confirming the specificity of the method.
For the typing, the T-category transitions of each strain are compared with the transitions of the other strains considered as reference strains. In practice, a value 0 is assigned when the transitions between the two strains are classified in the same category (positive or negative) and a value 1 is assigned when the transitions between the two strains are classified in different categories (a positive transition and a negative transition). The values are summed for all the T-category transitions of each strain pair in order to establish a score. The scores are shown in TABLE 6:
TABLE 6:
I I |iD2|iD3 ID4|AST ASTIASTIAST|VIR|VIR|VIR| IASTIVIR VIR
1U2 lUj im ^ ^ ^ ^ 8 13 14 5 6 7 ^^' 2 21 26
ID2 "o~ 17 6 1 17 7 24 24 ~IQ" 18 "si iTITIi 51 sFIF
IDS 17 0 15_J8 0 16 15 15 27 25 42 9 29 43 46 43 43
ID4 6 15 0 7 15 3 30 30 14 24 55 22 42 58 57 58 58
ID2a 1 18 7 0 18 8 23 23 9 17 50 17 37 51 50 51 51_
ID3a 17 0 15_J8 0 16 15 15 27 25 42 9 29 43 46 43 43
ID4a 7 16_^ 8 16 0 31 31 15 25 54 23 41 59 58 59 59
AST7 24 15 30 23 15_^1 0 4 20 12 27 8 16 32 35 32 34
AST8 24 15 30 23 15__31__4 0 20 14 27 10 14 32 35 32 34
AST13 10 27 14 9 27 15 20 20 0 14 43 24 30 46 45 46 46
AST14 18 25 24 17 25 25 12 14 14 0 33 18 24 40 43 40 40
VIR5 51 42 55 50 42 54 27 27 43 33 0 35 13 27 28 25 27
VIR6 18 9_^J7 9_^3 8 10 24 18 35 0 24 40 43 40 42
VIR7 38 29 42 37 29 41 16 14 30 24 13 24 0 34 37 34 36
ID7 52 43 58 51 43 59 32 32 46 40 27 40 34 - - - -_
AST2 51 46 57 50 46 58 35 35 45 43 28 43 37 - - ^ -_
VIR21 52 43 58 51 43 59 32 32 46 40 25 40 34 - - - -_
VIR26 52 _43_ 58 51 _43_ 59 34 34 46 40 27 42 36 -

62
The strains which have a score less than or equal to 4 are of the same type, the strains which have a score strictly above 4 are of different type. Thus, the ID2 and ID2a, ID3 and ID3a, ID4 and ID4a and AST7 and AST8 strains are of the same type. All the other strains taken in pairs are of different type. The high sums obtained between the ID7, AST2, VIR21 and VIR26 strains, which are not S. aureus, and all the other strains which are S. aureus, should be noted. These results confirm the specificity of the method.
The ID7, AST2, VIR21 and VIR26 strains are not of course of the same type; this would be absurd, they are of different species. These strains cannot therefore be compared with one another and no value is reported in TABLE 6. Extremely advantageously, scores above 25, for instance between ID2 and VR7, reflect a great divergence between strains. Scores between 15 and 25, as between ID2 and AST14, reflect a moderate divergence and scores between 5 and 15, as between ID2 and ID4a, a weak divergence.
The method thus implemented therefore makes it possible not only to establish whether two strains are of the same type, which is important for identifying a common seat of infection, but also to estimate the proximity of two strains, which is extremely important for epidemiological studies.
This example shows that, very advantageously, the present invention makes it possible, in a time of less than one hour, which is very short, to confirm the identity of a species such as S. aureus and to determine, simultaneously within the same analysis, the properties of typing and potential resistance to at least one antibiotic and to establish the existence of a virulence factor. These properties were established with the same instrument, which greatly facilitates the analysis and the reporting of the results. Finally, the characteristics of the bacteria are established using bacterial proteins, which reflects the existence of live and viable microorganisms, unlike characterizations using bacterial DNA which can be distorted by the existence of dead bacteria.
Example 6: Protocol for digestion of microorganisms with a desalifying step
Conventionally, the following protocol is implemented in 17 steps: Steps 1 to 10: idem example 4.

63
11. The sample volume is made up to 1 ml with water/0.1% (v/v) formic acid.
12. Equilibration of Waters Oasis HLB columns with 1 ml of methanol then 1 ml of HiO/O.P/o (v/v) formic acid.
13. Loading of the sample which flows by gravity.
14. Washing with 1 ml of H2O/0.i% (v/v) formic acid.
15. Elution with 1 ml of a mixture of 80% methanol and 20% water/0.1% (v/v) formic acid.
16. The eluate is evaporated with a SpeedVac® SPD2010 evaporator (Thermo Electron Corporation, Waltham, Massachusetts, United States of America), for two hours, in order to obtain a volume of approximately 100 \x\.
17. The eluate is then taken up in a water/0.5% (v/v) formic acid solution, quantity sufficient for (QS) 250 \il.
Example 7: Characterization of E coli samples:
After having established the one or more species of the samples according to any one of the methods described in examples 1 to 3, the species listed below are analyzed.
Fifteen E coli strains are analyzed in order to confirm their identification and to establish their characteristics:
ASTl AST2
AST3 AST4
AST5 VIR41
VIR42 VIR43
VIR44 V1R45
ID6 ID7
ID8 ID9
IDIO
The same method of analysis is applied to species not belonging to the E. coli species in order to serve as a negative control:
• S. aureus VIRIO
• S. pneumoniae

64 VIR19
• C. difficile
VIR28
Each sample is treated according to example 6, then a volume of 5 |al of digested proteins is injected and analyzed according to the following conditions:
• Agilent 1100 series chromatographic system from the company Agilent Technologies (Massy, France).
• Waters Symmetry CI8 column, 2.1 mm internal diameter, 100 mm long, particle size 3.5 jam.
• Solvent A: H2O + 0.1 % formic acid.
• Solvents: ACN +0.1% formic acid.
• HPLC gradient defined in TABLE 7 hereinafter:
TABLE 7
Time(min) I Flow rate I Solvent A (%) I Solvent B(%)
(Ml)
0 300 95 5
3 300 95 5
28 300 60 40
30 300 0 100
38 300 0 100
38l 300 95 5
45 300 95 5
• The eluate at the output of the chromatographic column is directly injected into the ionization source of the QTRAP® 5500 mass spectrometer from the company Applied Biosystems (Foster City, United States of America).
• The peptides, resulting from the digestion of the proteins of the microorganism, are analyzed using the mass spectrometer in MRM mode. Only the peptides indicated in TABLE 8 are detected. For this, the first-generation fragment(s) indicated in TABLE 8 is (are) detected. The application, to which each

65 transition, i.e. each peptide associated with its first-generation fragment, makes it possible to respond, is specified in the clinical interest column of TABLE 8 with the letters I, T, R and V. I denotes the confirmation of the identification of
the microorganism, T the typing, R the resistance to at least one antibiotic and
V the detection of virulence factors.
TABLE 8:
Transition r> . • ^ .-j first-generation Clinical
, Protein Peptide i> ^ ■
number fragment ion interest
1 AC0N2~ ILEIEGLPDLK y7 singly charged I + T
2 AC0N2 ILEIEGLPDLK y8 singly charged I + T
3 AC0N2 ILEIEGLPDLK y9 singly charged I + T
4 AC0N2 VADGATWSTSTR y7 singly charged I + T
5 AC0N2 VADGATWSTSTR y8 singly charged I + T
6 AC0N2 VADGATWSTSTR y9 singly charged I + T
7 ASPG2 TNTTDVATFK y6 singly charged I + T
8 ASPG2 TNTTDVATFK y7 singly charged I + T
9 ASPG2 TNTTDVATFK y8 singly charged I + T

10 FABB LDTTGLIDR y6 singly charged I + T
11 FABB LDTTGLIDR y7 singly charged I + T
12 FABB LDTTGLIDR y8 singly charged I + T
13 FABB VGLL\GSGGGSPR y8 singly charged I + T
14 FABB VGLIAGSGGGSPR y9 singly charged I + T
15 FABB VGLIAGSGGGSPR ylO singly charged I + T
16 GLNH AIDFSDGYYK y6 singly charged I + T
17 GLNH AIDFSDGYYK y7 singly charged I + T
18 GLNH AIDFSDGYYK y8 singly charged I + T
19 MODA LGAWDTLSPK y7 singly charged I + T
20 MODA LGAWDTLSPK yS singly charged I + T
21 MODA LGAWDTLSPK y9 singly charged I + T

22 0DP2 FGEIEEVELGR y7 singly charged I + T
23 0DP2 FGEIEEVELGR y8 singly charged I + T
24 QDP2 FGEIEEVELGR y9 singly charged I + T
25 OMPC INLLDDNQFTR y7 singly charged I + T
26 OMPC INLLDDNQFTR y8 singly charged I + T
27 OMPC INLLDDNQFTR y9 singly charged I + T
28 PFLB LATAWEGFTK y6 singly charged I + T
29 PFLB LATAWEGFTK y7 singly charged I + T
30 PFLB LATAWEGFTK y8 singly charged I + T
31 SUCD DSILEAIDAGDC y8 singly charged I + T
32 SUCD DSILEAIDAGIK y9 singly charged I + T
33 SUCD DSILEAIDAGIK y 10 singly charged I + T
34 SUCD FAALEAAGVK y7 singly charged I + T
35 SUCD FAALEAAGVK y8 singly charged I + T
36 SUCD FAALEAAGVK y9 singly charged I + T
37 I SUCD I SLADIGEALK | y6 singly charged | I + T

66
38 I SUCD I SLADIGEALK I y7 singly charged I I + T
39 SUCD SLADIGEALK y8 singly charged I + T
40 TKTl TEEQLANIAR y7 singly charged I + T
41 TKTl TEEQLANIAR y8 singly charged I + T
42 TKTl TEEQLANIAR y9 singly charged I + T
43 YFCZ AEAEQTLAALTEK yS singly charged I + T
44 YFCZ AEAEQTLAALTEK y9 singly charged I + T
45 YFCZ AEAEQTLAALTEK ylO singly charged I + T
46 YGAU SGDTLSAISK y6 singly charged I + T
47 YGAU SGDTLSAISK y7 singly charged I + T
48 YGAU SGDTLSAISK y8 singly charged I + T
49 TEM-2 LLTGELLTLASR y7 singly charged R + T
50 TEM-2 LLTGELLTLASR y8 singly charged R + T
51 TEM-2 LLTGELLTLASR y9 singly charged R + T
52 TEM-2 SALPAGWFIADK y7 singly charged R + T
53 TEM-2 SALPAGWFLADK y8 singly charged R + T
54 TEM-2 SALPAGWFIADK y9 singly charged R + T
55 TEM-2 VAGPLLR y4 singly charged R + T
56 TEM-2 VAGPLLR y5 singly charged R + T
57 TEM-2 VAGPLLR y6 singly charged R + T
58 TEM-2 VGYIELDLNSGK y8 singly charged R + T
59 TEM-2 VGYIELDLNSGK y9 singly charged R + T
60 TEM-2 VGYIELDLNSGK y 10 singly charged R + T
61 TEM-2 VLLCGAVLSR y7 singly charged R + T
62 TEM-2 VLLCGAVLSR y8 singly charged R + T
63 TEM-2 VLLCGAVLSR y9 singly charged R + T
64 ASPA ISDIPEFVR y5 singly charged T
65 ASPA ISDIPEFVR y6 singly charged T
66 ASPA ISDIPEFVR y7 singly charged T
67 ASPA lEEDLLGTR y6 singly charged T
68 ASPA lEEDLLGTR y7 singly charged T
69 ASPA lEEDLLGTR y8 singly charged T
70 ASPA LVDAINQLR y6 singly charged T
71 ASPA LVDAINQLR y7 singly charged T
72 ASPA LVDAnS[QLR y8 singly charged T
73 ATPA TALAIDAIINQR y7 singly charged T
74 ATPA TALAIDAIINQR y8 singly charged T
75 ATPA TALAIDAIINQR y9 singly charged T
76 ATPA WNTLGAPIDGK y7 singly charged T
77 ATPA WNTLGAPIDGK y8 singly charged T
78 ATPA WNTLGAPIDGK y9 singly charged T

79 CHIP SAGGIVLTGSAAAK y9 singly charged T
80 CHIP SAGGIVLTGSAAAKylO singly charged T
81 CHIP SAGGIVLTGSAAAK y 11 singly charged T
82 CH6P AVTAAVEELK y6 singly charged T
83 CH60 AVTAAVEELK y7 singly charged T
84 CH60 AVTAAVEELK y8 singly charged T
85 DBHB ALDAIL\SVTESLK y8 singly charged T
86 DBHB ALDAIL\SVTESLK y9 singly charged T
87 I DBHB I ALDAIIASVTESLK I ylQ singly charged I T

67
88 I DCEB I YWDVELR I y5 singly charged I T
89 DCEB YWDVELR y6 singly charged T
90 DCEB YWDVELR y4 singly charged T
91 DHSA LPGILELSR y6 singly charged T
92 DHSA LPGILELSR y7 singly charged T
93 DHSA LPGILELSR y8 singly charged T

94 DPS SKATNLLYTR y6 singly charged T
95 DPS SKATNLLYTR y7 singly charged T
96 DPS SKATNLLYTR y8 singly charged T
97 HNS SEALKILNNIR y7 singly charged T
98 HNS SEALKILNNIR y8 singly charged T
99 HNS SEALKILNNIR y9 singly charged T
100 MDH LFGVTTLDIIR y6 singly charged T
101 MDH LFGVTTLDIIR y7 singly charged T
102 MDH LFGVTTLDIIR y8 singly charged T
103 PGK ASLPTIELALK y7 singly charged T
104 PGK ASLPTIELALK y8 singly charged T
105 PGK ASLPTIELALK y9 singly charged T
106 PUR7 LLSDTECLVK y6 singly charged T
107 PUR7 LLSDTECLVK y7 singly charged T
108 PUR7 LLSDTECLVK y8 singly charged T

109 RL4 SILSELVR y5 singly charged T
110 RL4 SILSELVR y6 singly charged T
111 RL4 SILSELVR y7 singly charged T
112 RSI GGFTVELNGIR y7 singly charged T
113 RSI GGFTVELNGIR y8 singly charged T
114 RSI GGFTVELNGIR y9 singly charged T

115 YJGF TGEVPADVAAQAR y8 singly charged T
116 YJGF TGEVPADVAAQAR y9 singly charged T
117 YJGF TGEVPADVAAQAR y 10 singly charged T
118 stxlA TYVDSLNVIR y6 singly charged V + T
119 stxlA TYVDSLNVIR y7 singly charged V + T
120 stxlA TYVDSLNVIR y8 singly charged V + T

121 stxlA-2A FVTVTAEALR y7 singly charged V + T
122 stxlA-2A FVTVTAEALR y8 singly charged V + T
123 stx2A ISNVLPEYR y6 singly charged V + T
•124 stx2A ISNVLPEYR y7 singly charged V + T
^125 I stx2A I ISNVLPEYR | y8 singly charged | V + T
The charge state of the precursor peptide, its retention time and the transitions, i.e. the
ratios (m/z)i in Ql and (m/z)2 in Q3, are indicated in TABLE 9. The collision energy
used to fragment the precursor ion is also indicated in TABLE 9.

68 TABLE 9:
rr. ■ ■ ^ T^ • (m/z) (m/z) ^ ... .
Transition Precursor Retention r-,; , . ^,, , . Collision
filtered in filtered m
number charge state time „. ^- energy
i 2 19.04~ 620.36 771.42 ~ 36
2 2 19.04 620.36 884.51 36
3 2 19.04 620.36 1013.55 36
4 2 8.93 632.33 749.42 37
5 2 8.93 632.33 850.46 37
6 2 8.93 632.33 921.5 37
7 2 9.99 549.28 680.36 32
8 2 9.99 549.28 781.41 32
9 2 9.99 549.28 882.46 32
10 2 12.89 502.27 674.38 30
11 . 2 12.89 502.27 775.43 30
12 2 12.89 502.27 890.46 30
13 2 10.63 564.31 674.32 33
14 2 10.63 564.31 745.36 33
15 2 10.63 564.31 858.44 33
16 2 14.12 589.77 732.32 34
17 2 14.12 589.77 879.39 34
18 2 14.12 589.77 994.42 34
19 2 15.09 544.29 846.44 32
20 2 15.09 544.29 917.47 32
21 2 15.09 544.29 974.49 32
22 2 15.61 639.32 831.42 37
23 2 15.61 639.32 944.5 37
24 2 15.61 639.32 1073.55 37
25 2 16.42 674.85 895.39 39
26 2 16.42 674.85 1008.47 39
27 2 16.42 674.85 1121.56 39
28 2 15.49 562.29 767.37 33
29 2 15.49 562.29 838.41 33
30 2 15.49 562.29 939.46 33
31 2 19.84 622.84 816.45 36
32 2 19.84 622.84 929.53 36
33 2 19.84 622.84 1042.61 36
34 2 13.35 488.78 687.4 29
35 2 13.35 488.78 758.44 29
36 2 13.35 488.78 829.48 29
37 2 15.56 508.78 630.38 30
38 2 15.56 508.78 745.41 30
39 2 15.56 508.78 816.45 30
40 2 11.4 572.8 785.46 34
41 2 11.4 572.8 914.51 34
42 2 11.4 572.8 1043.55 34
43 2 14.85 687.86 846.49 39
44 2 14.85 687.86 974.55 39
45 I 2 I 14.85 I 687.86 | 1103.59 | 39

69
46 I 2 I 9.8 I 489.76 I 618.38 I 29
47 2 9.8 489.76 719.43 29
48 2 9.8 489.76 834.46 29
49 2 19.38 643.89 773.49 33
50 2 19.38 643.89 902.53 33
51 2 19.38 643.89 959.55 33
52 2 18.43 638.34 836.43 33
53 2 18.43 638.34 907.47 33
54 2 18.43 638.34 1004.52 33
55 2 10.79 363.24 498.34 21
56 2 10.79 363.24 555.36 21
57 2 10.79 363.24 626.4 21
58 2 15.94 654.35 875.45 34
59 2 15.94 654.35 988.53 34
60 2 15.94 654.35 1151.59 34
61 2 14.8 544.32 762.39 29
62 2 14.8 544.32 875.48 29
63 2 14.8 544.32 988.56 29
64 2 12.8 538.29 647.35 29
65 2 12.8 538.29 760.44 29
66 2 12.8 538.29 875.46 29
67 2 12.9 523.28 674.38 28
68 2 12.9 523.28 803.43 28
69 2 12.9 523.28 932.47 28
70 2 15.31 521.31 714.43 28
71 2 15.31 521.31 829.45 28
72 2 15.31 521.31 928.52 28
73 2 17.37 649.87 829.45 34
74 2 17.37 649.87 942.54 34
75 2 17.37 649.87 1013.57 34
76 2 11.94 592.34 657.36 31
77 2 11.94 592.34 770.44 31
78 2 11.94 592.34 871.49 31
79 2 11.08 601.84 817.48 31
80 2 11.08 601.84 930.56 31
81 2 11.08 601.84 987.58 31
82 2 11.86 515.79 688.39 28
83 2 11.86 515.79 759.42 28
84 2 11.86 515.79 860.47 28
85 2 22.24 715.91 834.46 36
86 2 22.24 715.91 947.54 36
87 2 22.24 715.91 1060.62 36
88 2 15.35 490.75 631.34 27
89 2 15.35 490.75 817.42 27
90 2 15.35 490.75 516.31 27
91 2 17.1 499.31 730.45 27
92 2 17.1 499.31 787.47 27
93 2 17.1 499.31 884.52 27
94 2 10.02 583.83 779.44 31
95 I 2 I 10.02 I 583.83 | 880.49 | 31

70
96 I 2 I 10.02 I 583.83 I 951.53 I 31
97 2 14.94 635.88 870.55 33
98 2 14.94 635.88 983.64 33
99 2 14.94 635.88 1054.67 33
100 2 20.34 624.37 730.45 32
101 2 20.34 624.37 831.49 32
102 2 20.34 624.37 930.56 32
103 2 18.16 578.35 787.49 30
104 2 18.16 578.35 884.55 30
105 2 18.16 578.35 997.63 30
106 2 13.11 589.31 749.39 31
107 2 13.11 589.31 864.41 31
108 2 13.11 589.31 951.45 31
109 2 17.68 458.78 603.35 25
110 2 17.68 458.78 716.43 25
111 2 17.68 458.78 829.51 25
112 2 15.99 581.81 800.46 31
113 2 15.99 581.81 901.51 31
114 2 15.99 581.81 1048.58 31
115 2 10.34 642.83 801.42 33
116 2 10.34 642.83 898.47 33
117 2 10.34 642.83 997.54 33
118 2 14.85 590.32 701.43 31
119 2 14.85 590.32 816.46 31
120 2 14.85 590.32 915.53 31
121 2 14.15 553.81 759.44 29
122 2 14.15 553.81 860.48 29
123 2 12.9 545.8 776.43 29
124 2 12.9 545.8 890.47 29
~l25 I 2 I 12.9 I 545.8 | 977.51 | 29
• The other machine parameters used are the following:
Scan type: MRM
Scheduled MRM: yes
Polarity: Positive
Ionization source: Turbo V™ (Applied BioSystems)
Ql setting: Filtering with unit resolution
Q3 setting: Filtering with unit resolution
Inter-scan pause: 5.00 msec
Scan speed: lODa/s
Curtain gas: 5 0.00 psi
Cone voltage: 5500.00 V
Source temperature: 550.00°C

71
Nebulizing gas: 50.00 psi
Heating gas: 40.00 psi
Dynamic filling: activated
Declustering potential (DP): 100.00 V
Entry potential before QO (EP): 9.00 V
Collision cell exit potential (CXP): 35 V
Total cycle time: 1.2 sec
Detection window 80 sec
The areas obtained for each of the transitions and for each of the microorganisms studied were measured. All the transitions with an area greater than or equal to 2500 (arbitrary units) are considered to be positive and have been denoted "1" in tables lOA and lOB. All the transitions with an area less than 2500 are considered to be negative and have been denoted 0 in tables lOA and lOB. When no signal peak was observed, the transition was noted as negative.
The positive-transition number is then summed for the 1, R and V applications and reported in TABLE 11:
TABLE lOA:
Transition ^^^^ ^^.^2 AST3 AST4 AST5 VIR41 VIR42 VIR43 VIR44
number
1 111111111
2 111111111
3 111111111
4 1 1 1 1 ^__^ 1 1 1
5 \ \_^^_i 1 1____J: 1 1 1
6 1 1 1 1 1^^ 1 1 1
7 1111 1^^ 1 1 1
8 1 1 1__^ 1^^ 1 1 1
9 1 1 1^^ ^^ 1 1 1
10 1 1 1 1 ^___J 1^_J 1
11 \ 1____J 1 ^^ 1 1 1
12 L__^ I 1 1 1 1 1 1
13 1 1___^ I l____i 1 1 1
14 1 1 1 1 1 1 1 1 1
15 1 1 ]__^ 1 1 1 1 1
16 111111111
17 I 1 I 1 I 1 I 1 I 1 I 1 I 1 I 1 I 1

72
18 I 1 I 1 I 1 I 1 I 1 I 1 I 1 I 1 I 1
19 111111111
20 111111111
21 111111111
22 111111111
23 111111111
24 111111111
25 111111111
26 111111111
27 1 1 1 110 0 0 0
28 111111111
29 111111111
30 111111111
31 111111111
32 111111111
33 111111111
34 111111111
35 111111111
36 111111111
37 111111111
38 1 1 1 1 1 1 1 1 1
39 111111111
40 11111 1 1 1 1
41 111111111
42 110 0 0 1111
43 111111111
44 L_^ 1 1^^ I I I 1
45 1 ^__J 1 1 1 1 1 1
46 111111111
47 111111111
48 111111111
49 0 11 1 ]____1 1 1 1
50 0 1 1 1 1 0 0 0 0
51 0 11111111
52 0 11110 0 11
53 0 1 1 1 1 0 0 0 0
54 0 1 1 1 1 0 0 0 0
55 0 11111111
56 1 1 1 1 1 0 0 0 1
57 0 1 1 1 1 0 0 0 0
58 0 1 1 1 1 0 0 0 0
59 0 1 1 1 1 0 0 0 0
60 0____J 1 1 10 0 0 0
61 0 1 1 1^ 1 0 0 0 0
62 0 11110 111
63 0 1 1 1 1 0 0 0 0
64 000000001
65 0 0 0 0 0 0 0 0 1
66 0 0 0 0 0 1 1 1 1
67 I 1 I 1 I 1 I 1 I 1 I 1 I 1 I 1 I 1

73
68 I 1 I 1 I 1 I 1 I 1 I 1 I 1 I 1 I 1
69 111111111
70 111111111
71 1 1 1 1^^ 1 ]____J 1
72 1 1 1 L__^^ 1 I I 1
73 111111111
74 111111111
75 111111111
76 111111111
77 111111111
78 111111111
79 111111111
80 111111111
81 1 1 1 1____J 1 1 1 1
82 11111 1 1 1 1
83 1 1 1 ^^ 1 1 1 1
84 111111111
85 1 1 1 ^_J 1 1 1 1
86 111111111
87 111111111
88 111111111
89 0 11111111
90 111111111
91 111111111
92 111111111
93 111111111
94 1 1 1 1^^ 1 1 1 0
95 111111110
96 I 1^__J 1 1 1 1 1 1
97 0 0 0 0 0 1 1 1 1
98 0 0 0 0 0 1 1 1 0
99 000001000
100 111111111
101 111111111
102 111111111
103 111111111
104 111111111
105 1111 1 1 1 1 1
106 1^_J 1 1____J 1 1 1 1
107 ^^ 1 1 1 1 1 I 1
108 1 1 1 1 L___! I 1 1
109 1 1 1 1 1 1 1 1 1
110 111111111
111 1 1 1 1 1 1 1 1 1
112 1 ^^ 1 1 1 1 \ 1
113 1 \ 1 1 1 1 1 1 1
114 000000001
115 1 1 I l___^ I 1 1 1
L16 1 1 1 1 1 L_^ I L_
117 I 1 I 1 I 1 I 1 I 1 I 1 I 1 I 1 I 1

74
118 I 0 I 0 I 0 I 0 I 0 I 1 I 0 I 1 I 0
119 10 0 0 0 10 10
120 0 110 110 10
121 0 0 0 0 0 1 11 1
122 10 0 0 0 1 1 1 1
123 0 0 0 0 0 1 1 0 1
124 0 10 0 11111
~125 |o|o|o|o|o|l|l|o|l~
TABLE lOB:
Transition ^^^^ ^^^ jj^^ ^^ j^^ J^JQ ^^^^^ ^j^^g ^j^^
number
1 1111110 0 0
2 1111110 0 0
3 1111 1 10 0 0
4 1111110 0 0
5 1111110 0 0
6 1____J 1^^ 1 10 0 0
7 1 1 1 1 1 10 0 0
8 1111110 0 0
9 1111110 0 0
10 1 1 1 1 1 10 0 0
11 1111110 0 1
12 1 1 1 1 1 10 0 0
13 1111110 0 0
14 1111110 0 1
15 1 1 1 1 1 10 0 0
16 1 1 1 1 1 10 0 0
17 1111110 0 0
18 1111110 0 0
19 1111110 0 0
20 1 1^^ 1 1 10 0 0
21 1111110 0 0
22 1 1 1 1 1 1 0 0 0
23 1 1 1 1 1 10 0 0
24 1111110 0 0
25 1111110 0 0
26 1111 1 10 0 0
27 1 1 1 1 1 1 0 0 0
28 1 1 1 1 1 1 0 0 0
29 1 1 1 1 1 1 0 0 0
30 1 1 1 1 1 1 0 0 0
31 1111 1 10 0 0
32 1 1 1 1 1 10 0 0
33 1 10 0 0 0 0 0 0
34 1 1 1 1 1 \ 1 0 0
35 1 1 1 1 1 10 10
~ 36 I 1 I 1 I 1 I 1 I 1 I 1 I 0 I 0 I 0

75
37 I 1 I 1 I 1 I 1 I 1 I 1 I 0 I 0 I 0
38 1 1__^ 1^^ 10 0 0
39 1 1 1 1 1 10 0 0
40 1 1 1 1 1 1 0 0 0
41 1 1 1 1 1 1 0 0 0
42 1 1 0 0 0 0 0 0 0
• 43 1 1 1 1 1 1 0 0 0
44 1 1 1___^ 1 1 OOP
45 1 10 0 0 0 0 0 0
46 1 1 1____1 1 1 0 0 0
47 1 1 1 1 1 1 0 0 0
48 1 1 1 1 1 1 0 0 0
49 1 00000000
50 000000000
51 100000000
52 100000000
53 000000010
54 000000000
55 1 00000000
56 000000000
57 000000000
58 0 0 0 0 1 1 1 0 0
59 000000000
60 0 0 0 0 00 0 0 0
61 0 0 1110 0 0 0
62 1 0 1 1 10 0 0 0
63 000000000
64 100000000
65 100000000
66 100000000
67 1111110 0 0
68 11111110 0
69 1 1 1 ]___^^ 10 10
70 1 1111 10 0 0
71 1 1^^ 1 1 10 0 0
72 1 1 0 0 0 0 0 0 0
73 1 1 1 1 1 1 0 0 0
74 1111110 0 0
75 1111110 0 0
76 1 1111 10 0 0
77 1 1 1 1 1 1 OOP
78 1 1 1 1 1 1 0 0 P
79 11111 1 1 1 P
8P 11111 10 0 0
81 1 1 1 1 1 1 P P P
82 1 1 1___J 1 1 POO
83 1111110 0 0
84 1 1 1 1 1 1 POO
85 1 1 1 1 1 1 0 0 P
86 I 1 I 1 I 1 I 1 I 1 I 1 I 0 I 0 I 0

76
87 |I|I|O|O|O|O|O|Q|O
88 1 0 1 1 1 10 0 0
89 10 11110 0 0
90 1 1 1 1 1 1 OOP
91 11111110 0
92 1 1 1 1 1 1 OOP
93 1 1 1 1 1 10 0 0
94 0__^ ! I 1 I 0 0 0
95 1 1 1 1 1 1 0 P 0
96 1 1 1 1 1 1 P P 0
97 1 1 1 1 1 1 P P 0
98 1 P 1 0 1 0 0 P P
99 OOPOOOOOO
100 1 1 1 1 1 1 P P P
101 1 1 1 1 1^__1 1 0 0
102 1 1 1 1 1 10 0 0
103 1111 1 10 0 0
104 1 1 1 1 1 1 P 0 P
1P5 1 1 1 1 1 1 P 0 0
1P6 1 1 1 1 1 1 P 0 0
1P7 \ 1 I 1 1 1 P P P
1P8 \ 1 1 1 1 1 P P P
1P9 1 1 1 1 1 1 P 0 P
IIP 1 1 1 1 1 1 P P P
111 1 1 1 1 1 1 P P P
112 1 1 1 1 1 1 0 0 P
113 1 1 1 1 1 1 0 0 P
114 1 PPOOOOPP
115 1 1 1 1 1 1 P P 0
116 1 1 1 1 1 1 P P P
117 1111 1 1 P P P
118 OOOOOOOPO
119 000000000
120 000000000
121 OOOPOOOOO
122 0^__^ 0 1 1 1 0 0 0
123 1 00000000
124 lOOPPPPPP
~125 |p|p|p|p|o|o|o|o|o~
TABLE 11:
c,, . p • Transitions Transitions Transitions
Strains Species ^ _
1 K V
ASTl E. coli 48 ' 1 ~ 2
AST2 E. coli 48 15 2
AST3 E. coli 47 15 1
AST4 E. coli 47 15 0
AST5 E. coli 47 15 2
"VIR41 I E.coli I 47 I 3 I 8

77
VIR42 I E. coli I 47 I 4 I 5
VIR43 E. coli 47 5 6
VIR44 E. coli 47 6 5
VIR45 E. coli 48 5 2
ID6 E. coli 48 0 1
ID7 E. coli 45 2 0
IDS E. coli 45 2 1
ID9 E. coli 45 3 1
ID 10 E. coli 45 1 1
VIRIO S. aureus 1 1 0
VIR19 S. pneumoniae 1 1 0
" VIR28 I C. difficile | 2 | 0 | 0
All the E. coli samples exhibit more than 44 positive transitions in the I category. All these samples are therefore confirmed as indeed belonging to the E. coli species.
On the other hand, the VIRIO, VIR19 and VIR28 samples exhibit less than 3 positive transitions in the I category, these samples are therefore confirmed as not belonging to the E. coli species.
The AST2, AST3, AST4 and AST5 strains of E. coli exhibit more than 14 positive transitions for the R category, they therefore express the plasmid-mediated penicillinase TEM-2, this being synonymous with a mechanism of resistance to penicillins.
On the other hand, the ASTl, VIR41, VIR42, VIR43, VIR44, VIR45, ID6, ID7, IDS, ID9 and IDIO strains of E. coli exhibit less than 7 positive transitions for the R category, they do not therefore express the plasmid-mediated penicillinase TEM-2. These strains are therefore sensitive to penicillins, in particular aminopenicillins or A penicillins (ampicillin), carboxypenicillins or C penicillins (ticarcillin) and ureidopenicillin or penicillin U (piperacillin).
The VIRIO, VIR19 and VIR28 strains which do not belong to the E. coli species exhibit less than two transitions for the R category, they do not therefore express TEM-2, thereby confirming the specificity of the method.
The VIR41, VIR42, VIR43 and VIR44 samples of £. coli exhibit more than four positive transitions in the V category, they therefore express the shigatoxin 1 or 2 (STXl or STX2) toxins. More specifically, for VIR41, transitions 118 to 125 are

78 positive, VIR41 therefore simultaneously expresses shigatoxin 1 and shigatoxin 2. VIR42 and VIR44 are positive for transitions 121 to 125, they therefore express shigatoxin 2. The same is true for VIR45, which exhibits transitions 123 and 124. VIR43, which exhibits transitions 118 to 122, expresses shigatoxin 2. On the other hand, the ASTl, AST2, AST3, AST4, AST5, VIR45, ID6, ID7, IDS, ID9 and IDIO strains of E. coli exhibit less than three positive transitions, they do not therefore express shigatoxin. These strains do not therefore have the properties of virulence related to shigatoxins.
The VIRIO, VIR19 and VIR28 strains which do not belong to the E. coli species also exhibit less than three transitions for the V category, they do not therefore express stxl or stx2, thereby confirming the specificity of the method.
For the typing, the T-category transitions of each strain are compared with the transitions of the other strains considered as reference strains. In practice, a value 0 is assigned when the transitions between the two strains are classified in the same category (positive or negative) and a value of 1 is assigned when the transitions between the two strains are classified in different categories (a positive transition and a negative transition). The values are summed for all the T-category transitions of each strain pair in order to establish a score. The scores are given in TABLE 12:
TABLE 12;
AST! AST2 AST3 AST4 AST5 VIR41 VIR42 VIR43 VIR44VIR45ID6 ID7 IDS ID9 IDIO VIRIO VIR19 VIR28
ASTl 0 19 18 18 19 17 12 15 17 16 3 U 12__n 9 92 92 95
AST2 19 0 3 3 2 24 21 18 22 19 22 22 23 22 22 109 109 112
AST3 18 3 0 0 1 25 22 21 23 20 19 19 20 19 19 106 106 109
AST4 18 3 0 0 1 25 22 21 23 20 19 19 20 19 19 106 106 109
AST5 19 2 1 1 0 24 23 20 24 21 20 20 21 20 20 107 107 110
VIR41 17 24 25 25 24 0 5 6 12 13 16 22 23 20 20 105 105 108
VIR42 12 21 22 22 23 5 0 7 7 8 13 19 20 17 17 102 102 105
VIR43 15 18 21 21 20 6 7 0 10 9 16 20 21 18 20 105 105 108
VIR44 17 22 23 23 24 12 7 10 0 5 18 22 23 22 22 103 103 106
VIR45 16 19 20 20 21 13 8 9 5 0 17 19 20 19 19 102 102 105
ID6 3 22 19 19 20 16 13 16 18 17 0 10_J1 10 8 91 91 94
ID7 11 22 19 19 20 22 19 20 22 19 10 0 1 4 4 89 89 92
ID8 12 23 20 20 21 23 20 21 23 20 11 1 0 2 3 90 92 92
ID9 11 22 19 19 20 20 17 18 22 19 10 4 2 0 3 90 94 94
IDIO I 9 I 22 I 19 I 19 I 20 I 20 I 17 I 20 I 22 I 19 | 8 4 3 3 0 87 91 91

79
VIRIO 92 109 106 106 107 105 102 105 103 102 91 89 90 90 87 ~ VIR19 92 109 106 106 107 105 102 105 103 102 91 89 92 94 91 ~ V1R28| 95 I 112 I 109 | 109 | 110 | 108 | 105 | 108 [ 106 | 105 | 941 92 | 92 | 94 | 91 | ~ | ~ | --
The strains which have a score less than or equal to 4 are of the same type, the strains which have a score strictly greater than 4 are of different type. Thus, the AST2, AST3, AST4 and AST5 strains are of the same type. The same is true, respectively, for the AST! and ID6 strains and for the ID7, IDS, ID9 and IDIO strains. All the other strains taken in pairs are of different types. The high sums obtained between the VIRIO, VIR19 and VIR28 strains, which are not S. aureus, and all the other strains which are S. aureus, should be noted. These results confirm the specificity of the method.
The VIRIO, VIR19 and VIR28 strains are of different species. These strains cannot therefore be compared with one another and no value is reported in TABLE 12. Extremely advantageously, scores greater than 20, for instance between AST4 and VIR41, reflect a great divergence between strains. Scores between 12 and 20, as between ID7 and VIR42, reflect a moderate divergence and scores between 4 and 12, as between IDIO and ASTl, a weak divergence.
The method thus implemented therefore makes it possible not only to establish whether two strains are of the same type, which is important for identifying a common seat of infection, but also to estimate the proximity of two strains, which is extremely important for epidemiological studies.
This example shows that, very advantageously, the present invention makes it possible, in a time of less than one hour, which is very short, to confirm the identity of a species such as E. coli and to determine, simultaneously within the same analysis, the properties of typing and potential resistance to at least one antibiotic and to establish the existence of a virulence factor. These properties were established with the same instrument, which greatly facilitates the analysis and the reporting of the results. Finally, the characteristics of the bacteria are established using bacterial proteins, which reflects the existence of live and viable microorganisms, unlike characterizations using bacterial DNA which can be distorted by the existence of dead bacteria.

80 Example 8; Protocol for digestion of microorganisms in the presence of methanol
Conventionally, the following protocol is implemented in 17 steps:
1. Sampling of a microorganism colony, obtained according to example 1, and suspension in 400 i^l of a 50 mM ammonium bicarbonate solution, pH=8.0.
2. Addition of 600 |j,l of methanol.
Steps 3 to 7: idem steps 2 to 6 of example 4. Steps 8 to 17: idem steps 8 to 17 of example 6.
Example 9: Characterization of C albicans samples:
After having established the one or more species of the samples according to any one of the methods described in examples 1 to 3, the species listed below are analyzed.
Seventeen C. albicans strains are analyzed in order to confirm their identification and to establish their characteristics:
ATFl ATF2
ATF3 ATF4
ATF5 ATF6
ATF7 VIR31
VIR32 VIR33
VIR34 VIR35
VIR36 VIR37
VIR38 VIR39
CA16
The same method of analysis is applied to species not belonging to the C. albicans species in order to serve as a negative control:
• E. coli VIR43
• S. aureus VIR5
• E. faecium AST-VAN8

81 Each sample is treated according to example 8, then a volume of 5 |al of digested proteins is injected and analyzed according to the same conditions as in example 7. • The peptides, resulting from the digestion of the proteins of the microorganism, are analyzed using the mass spectrometer in MRM mode. Only the peptides indicated in TABLE 13 are detected. For this, the first-generation fragment(s) indicated in TABLE 13 is (are) detected. The application, to which each transition, i.e. each peptide associated with its first-generation fragment, makes it possible to respond, is specified in the clinical interest column of TABLE 14 with the letters I, T, R and V. I denotes the confirmation of the identification of the microorganism, T the typing, R the resistance to at least one antibiotic and V the detection of virulence factors.
TABLE 13:
Transition „ , . r. *-j First-generation Clinical
, Protein Peptide jy ^ ■
number fragment ion interest
1 ALF ADEEFFAK y5 singly charged I + T
2 ALF ADEEFFAK y6 singly charged I + T
3 ALF ADEEFFAK y7 singly charged I + T

4 HSP75 AFDDESVQK y6 singly charged I + T
5 HSP75 AFDDESVQK y7 singly charged I + T
6 HSP75 AFDDESVQK y8 singly charged I + T
7 ENOl AKIDVVDQAK y6 singly charged I + T
8 ENOl AKIDVVDQAK y7 singly charged I + T
9 ENOl AKIDVVDQAK yS singly charged I + T

10 RL13 AVEVPEQTAYR y6 singly charged I + T
11 RL13 AVEVPEQTAYR y7 singly charged I + T
12 RL13 AVEVPEQTAYR y8 singly charged I + T
13 RS22 CGVIQPR y5 singly charged 1 + T
14 RS22 CGVIQPR y6 singly charged I + T
15 RS22 CGVIQPR y4 singly charged I + T
16 RLA4 DLQELIAEGNTK y8 singly charged I + T
17 RLA4 DLQELIAEGNTK y9 singly charged I + T
18 RLA4 DLQELIAEGNTK y7 singly charged I + T
19 PPIA FADENFVKR y6 singly charged I + T
20 PPIA FADENFVKR y7 singly charged I + T
21 PPIA FADENFVKR y8 singly charged I + T
22 TPIS FALDTGVK y6 singly charged I + T
23 TPIS FALDTGVK y7 singly charged I + T
24 TPIS FALDTGVK y5 singly charged I + T
25 RL28 GRLPEVPVrVK y8 singly charged I + T
26 RL28 GRLPEVPVrVK y5 singly charged 1 + T
27 I ALF I lAEALDIFHTK | y6 singly charged | I + T

82
28 I ALF I lAEALDIFHTK I y7 singly charged I I + T
29 ALP lAEALDIFHTK y8 singly charged I + T
30 ENOl IDVVDQAK y5 singly charged I + T
31 ENOl IDVVDQAK y6 singly charged I + T
32 ENOl IDVVDQAK y7 singly charged I + T
33 ENOl lEEELGSEAIYAGKDFQK yS singly charged I + T
34 ENOl lEEELGSEAIYAGKDFQK y9 singly charged I + T
35 PPIA lESFGSGSGATSK y9 singly charged I + T
36 PPIA lESFGSGSGATSK ylO singly charged I + T
37 PPIA lESFGSGSGATSK y 11 singly charged I + T
38 NTF2 LASLPFQK y5 singly charged I + T
39 NTF2 LASLPFQK y6 singly charged I + T
40 NTF2 LASLPFQK y7 singly charged I + T
41 MBFl LDATDDWAVK y7 singly charged I + T
42 MBFl LDATDDWAVK y8 singly charged I + T
43 MBFl LDATDDWAVK y9 singly charged I + T

44 PGK NVEHLVEK y6 singly charged I + T
45 PGK NVEHLVEK y7 singly charged I + T
46 PGK NVEHLVEK y5 singly charged I + T
47 RL36 SGIAAGVNK y5 singly charged I + T
48 RL36 SGIAAGVNK y6 singly charged I + T
49 RL36 SGIAAGVNK y7 singly charged I + T

50 G3P SGVDYVIESTGVFTK y9 singly charged I + T
51 G3P SGVDYVIESTGVFTK y 10 singly charged I + T
52 G3P SGVDYVIESTGVFTK yS singly charged I + T

53 EFIB SLNEFLADK y6 singly charged I + T
54 EFIB SLNEFLADK y7 singly charged I + T
55 RL13 SQETFDANVAR y7 singly charged I + T
56 RL13 SQETFDANVAR b8 singly charged I + T
57 RL13 SQETFDANVAR y9 singly charged I + T
58 CSlll SSSSTTKK y6 singly charged I + T
59 CSlll SSSSTTKK y7 singly charged I + T
60 HSP71 STLDPVGK y6 singly charged I + T
61 HSP71 STLDPVGK y7 singly charged I + T
62 HSP71 STLDPVGK y5 singly charged I + T

63 PGK SVELFQQAVAK y7 singly charged I + T
64 PGK SVELFQQAVAK y8 singly charged I + T
65 PGK SVELFQQAVAK y6 singly charged I + T

66 KPYK TANDVLELR y5 singly charged I + T
67 KPYK TANDVLELR y6 singly charged I + T
68 KPYK TANDVLELR y7 singly charged I + T
69 ENOl VGDKIQIVGDDLTVTNPTR y7 singly charged I + T
70 ENOl VGDKIQIVGDDLTVTNPTR y8 singly charged I + T
71 ENOl VGDKIQIVGDDLTVTNPTR y9 singly charged I + T
72 ADHl WAIDGGDEK y7 singly charged I + T
73 ADHl WAIDGGDEK y8 singly charged I + T
74 ADHl WAIDGGDEK y9 singly charged I + T
75 RS22 WTDNLLPAR y6 singly charged I + T
76 RS22 WTDNLLPAR y7 singly charged I + T
77 I RS22 I WTDNLLPAR | yS singly charged | I + T

83
78 I G3P |YKGEVTASGDDLVIDGHK|ylOsinglycharged| I + T
79 G3P YKGEVTASGDDLVIDGHK y 11 singly charged I + T
80 G3P YKGEVTASGDDLVIDGHK y9 singly charged I + T

81 ADHl YVLDTSK y5 singly charged I + T
82 ADHl YVLDTSK y6 singly charged I + T
83 ADHl YVLDTSK y4 singly charged I + T
84 CP51 AVIYDCPNSR y6 singly charged R + T
85 CP51 AVIYDCPNSR y7 singly charged R + T

86 CP51 GHYVLVFPGYAHTSER 10 singly charged R + T
87 CP51 GHYVLVFPGYAHTSER y9 singly charged R + T
88 CP51 GHYVLVSPGYAHTSER y9 singly charged R + T
89 CP51 GHYVLVSPGYAHTSER 10 singly charged R + T
90 CP51 GVIYDCPNSR y6 singly charged R + T
91 CP51 GVIYDCPNSR y7 singly charged R + T
92 CP51 GVIYDCPNSR y8 singly charged R + T
93 CP51 GVSSPYLPFGGGR y9 singly charged R + T
94 CP51 GVSSPYLPFGGGR y6 singly charged R + T
95 CP51 GVSSPYLPFSGGK y9 doubly charged R + T
96 CP51 GVSSPYLPFSGGK y9 singly charged R + T
97 CP51 GVSSPYLPFSGGK y6 singly charged R + T
98 CP51 GVSSPYLPFSGGR y9 doubly charged R + T
99 CP51 GVSSPYLPFSGGR y9 singly charged R + T
100 CP51 GVSSPYLPFSGGR y6 singly charged R + T

101 LIPASES AAVGDILQSR y7 singly charged V + T
102 LIPASE8 AAVGDILQSR y8 singly charged V + T
103 LIPASE8 ITPDDLR y5 singly charged V + T
104 LIPASES ITPDDLR y6 singly charged V + T
105 LIPASES ITPDDLR y4 singly charged V + T
106 LIPASES TGWDILK y5 singly charged V + T
107 LIPASES TGWDILK y6 singly charged V + T
IPS ILIPASESI TGWDILK | y4 singly charged | V + T
The charge state of the precursor peptide, its retention time and the transitions, i.e. the ratios (ni/z)i in Ql and (m/z)2 in Q3, are indicated in TABLE 14. The collision energy used to fragment the precursor ion is also indicated in TABLE 14.
TABLE 14:
Transition , Retention },^ j },^ ', Collision
, charge ^. filtered filtered
number . ° time . „, . _. energy
state m Ql m Q3 ^^
1 2 11.9 478.72 641.33 26
2 2 11.9 478.72 770.37 26
3 2 11.9 478.72 885.4 26
4 2 7.8 519.74 705.34 28
5 2 7.8 519.74 820.37 28
6 2 7.8 519.74 967.44 28
7 1 2 I 8.5 1543.811 659.37 I 29

84
8 I 2 I 8.5 1543.811 774.4 I 29
9 2 8.5 543.81 887.48 29
10 2 11 631.82 767.37 33
11 2 11 631.82 864.42 33
12 2 11 631.82 963.49 33
13 2 6.4 415.22 612.38 23
14 2 6.4 415.22 669.4 23
15 2 6.4 415.22 513.31 23.3
16 2 15.5 665.85 845.47 34
17 2 15.5 665.85 974.52 34
18 2 15.5 665.85 732.39 34.3
19 2 10.5 563.29 792.44 30
20 2 10.5 563.29 907.46 30
21 2 10.5 563.29 978.5 30
22 2 12.5 425.74 632.36 24
23 2 12.5 425.74 703.4 24
24 2 12.5 425.74 519.28 23.7
25 2 14.8 603.88 880.55 32
26 2 14.8 603.88 555.39 31.6
27 2 17.8 629.35 760.4 33
28 2 17.8 629.35 873.48 33
29 2 17.8 629.35 944.52 33
30 2 9 444.25 560.3 25
31 2 9 444.25 659.37 25
32 2 9 444.25 774.4 25
33 3 18.6 676.34 956.48 38
34 3 18.6 676.34 1069.57 38
35 2 8.4 614.3 751.36 32
36 2 8.4 614.3 898.43 32
37 2 8.4 614.3 985.46 32
38 2 14.1 452.27 632.38 25
39 2 14.1 452.27 719.41 25
40 2 14.1 452.27 790.45 25
41 2 12.1 573.31 745.41 30
42 2 12.1 573.31 846.46 30
43 2 12.1 573.31 917.49 30
44 2 7 484.26 754.41 26
45 2 7 484.26 853.48 26
46 2 7 484.26 625.37 26.3
47 2 4.5 408.73 488.28 23
48 2 4.5 408.73 559.32 23
49 2 4.5 408.73 672.4 23
50 2 18 801.41 981.53 40
51 2 18 801.41 1080.59 40
52 2 18 801.41 868.44 40.3
53 2 14.3 518.77 722.37 28
54 2 14.3 518.77 836.41 28
55 2 10.1 619.29 792.4 32
56 2 10.1 619.29 893.36 32
57 I 2 I 10.1 1619.2911022.491 32

85
58 I 2 I 10.6 1413.221 651.37 I 23
59 2 10.6 413.22 738.4 23
60 2 8.7 408.73 628.37 23
61 2 8.7 408.73 729.41 23
62 2 8.7 408.73 515.28 23
63 2 14.6 610.34 791.44 32
64 2 14.6 610.34 904.53 32
65 2 14.6 610.34 644.37 31.9
66 2 12.9 515.78 629.4 28
67 2 12.9 515.78 744.43 28
68 2 12.9 515.78 858.47 28
69 3 15.3 681.04 788.43 38
70 3 15.3 681.04 901.51 38
71 3 15.3 681.04 1016.54 38
72 2 7.7 501.76 733.34 27
73 2 7.7 501.76 804.37 27
74 2 7.7 501.76 903.44 27
75 2 14.9 543.29 683.42 29
76 2 14.9 543.29 798.45 29
77 2 14.9 543.29 899.49 29
78 2 11.4 952.47 1068.53 47
79 2 11.4 952.47 1155.56 47
80 2 11.4 952.47 1011.51 46.9
81 2 8.5 413.22 563.3 23
82 2 8.5 413.22 662.37 23
83 2 8.5 413.22 450.22 23.2
84 2 9.3 597.78 748.3 31
85 2 9.3 597.78 911.37 31
86 2 14.3 916.96 1164.54 45
87 2 14.3 916.96 1017.47 45.3
88 2 11 886.94 1017.47 44
89 2 11 886.94 1104.51 44
90 2 9.4 590.77 748.3 31
91 2 9.4 590.77 911.37 31
92 2 9.4 590.77 1024.45 31
93 2 16.1 647.33 963.5 33
94 2 16.1 647.33 590.3 33.5
95 2 15.8 648.34 483.26 34
96 2 15.8 648.34 965.51 34
97 2 15.8 648.34 592.31 33.5
98 2 16 662.34 497.26 34
99 2 16 662.34 993.52 34
100 2 16 662.34 620.32 34.1
101 2 12.75 515.29 788.43 28
102 2 12.75 515.29 887.49 28
103 2 9.83 415.22 615.31 23
104 2 9.83 415.22 716.36 23
105 2 9.83 415.22 518.26 23.3
106 2 15.81 416.73 674.39 23
107 I 2 I 15.81 1416.731 731.41 | 23

86 I 108 I 2 I 15.81 1416.731 488.31 | 23.3 |
The other machine parameters used are the same as in example 7. The areas obtained for each of the transitions and for each of the microorganisms studied were measured. All the transitions with an area greater than or equal to 2000 (arbitrary units) are considered to be positive and have been denoted "1" in tables ISA and 15B. All the transitions with an area less than 2000 are considered to be negative and have been denoted 0 in tables 15A and 15B. When no signal peak was observed, the transition was noted as negative.
TABLE ISA:
Transition ^^^^ ^^P2 ATF3 ATF4 VIR31 ATF5 ATF6 ATF7 VIR32 VIR33
number
1 1111111111
2 1111111111
3 1111111111
4 1111111111
5 1111111111
6 1111111111
7 1111111111
8 1111111111
9 1111111111
10 11110 1110 0
11 11110 1110 1
12 11110 1110 0
13 11110 11111
14 11110 11111
15 1111111111
16 1111111111
17 1111111111
18 1111111111
19 11110 1110 1
20 11110 11111
21 11110 1110 1
22 1111111111
23 1111111111
24 1111111111
25 1111111111
26 1111111111
27 0000100011
28 0000100011
29 000000000 1
30 1111111111
31 I 1 I 1 I 1 I 1 I 1 I 1 I 1 I 1 I 1 I 1

87
32 I 1 I 1 I 1 I 1 I 1 I 1 I 1 I 1 I 1 I 1
33 0000100011
34 0000000001
35 1111 111111
36 1111 111111
37 1111111111
38 11110 1110 0
39 11110 1110 1
40 11110 1110 0
41 1111111111
42 1111111111
43 .1 1 1 1 1 1 1 1 1 1
44 " 'l 1 1 1 1 1 1 1 1 1
45 1111111111
46 1111111111
47 1111111111
48 1111111111
49 00001000 11
50 1111111111
51 1111111111
52 1111111111
53 11110 11111
54 1111111111
55 11110 1110 0
56 11110 1110 0
57 11110 1110 0
58 0000 10001 1
59 0000100011
60 1111111111
61 11110 1110 1
62 1111111111
63 1111111111
64 11110 1110 0
65 11110 11111
66 1111111111
67 1111111111
68 1111111111
69 1111111111
70 1111111111
71 11110 11111
72 1111111111
73 1111111111
74 1111111111
75 1111111111
76 1111111111
77 1111111111
78 1111111111
79 1111111111
80 11110 11111
81 1111111111
82 1111111111
I 1 1 1 1 1 1 t I I -^ I I ^

88
83 I 1 I 1 I 1 I 1 I 1 I 1 I 1 I 1 I 1 I 1
84 0000001100
85 0000001100
86 0010000000
87 0110000000
88 0001000000
89 0001000000
90 110 10 110 0 1
91 110 10 110 0 0
92 0001000000
93 11110 0 10 0 0
94 11110 0 110 0
95 0 0 0 0 110 0 11
96 0 0 00010000
97 10 0 1110 111
98 0000000101
99 0000000100
100 00000001 00
101 0000100000
102 0000100000
103 0 0 0 0 10 0 0 1 1
104 0 0 0 110 0 0 1 1
105 0000100010
106 0000100010
107 0000100010
108 |o|o|o|o|l|o|o|o|l|o
TABLE 15B:
Transition ^^^^^ ^^^^ ^^^^^ ^^^^^ ^.^^^ ^^^^^ ^^^^ ^^^^ ^^^ AST-
number VAN8
1 11111110 0 0
2 11111110 0 0
3 11111110 0 0
4 11111110 0 0
5 11111110 0 0
6 11111110 0 0
7 111111110 0
8 11111110 0 0
9 11111110 0 0
10 0000001000
11 11111110 0 0
12 0000001100
13 111110 10 0 0
14 110 11110 0 0
15 11111110 0 0
16 11111110 0 0
17 11111110 0 0
18 11111110 0 0
19 I 1 I 1 I 1 I 1 I 1 I 1 I 1 I 0 I 0 I 0

89
20 I 1 I 1 I 1 I 1 I 1 I 1 I 1 I 1 I 0 I 0
21 11111110 0 0
22 11111110 0 0
23 111111110 0
24 111111110 0
25 1 1 1 1 1 1 10 0 0 .
26 11111110 0 0 27 1 1 1 1 1110 0 0

28 11111110 0 0
29 11111110 0 0
30 11111110 0 0
31 1 1 1 1 1 1 10 0 0
32 1111111111
33 11111110 0 0
34 110 11110 0 0
35 11111110 0 0
36 11111110 0 0
37 11111110 0 0
38 0000001000
39 1110 0 110 0 0
40 0000001000
41 11111110 0 0
42 11111110 0 0
43 11111110 0 0
44 11111110 0 0
45 11111110 0 0
46 11111110 0 0
47 11111110 0 0
48 11111110 0 0
49 1111110 0 0 0
50 11111110 0 0
51 0 1111110 0 0
52 11111110 0 0
53 11111110 0 0
54 11111110 0 0
55 0000001000
56 0000001000
57 0000001000
58 11111110 0 0
59 11111110 0 0
60 11111110 0 0 61 10 0 0 110 0 0 0

62 111111110 0
63 0 11111110 0
64 0 1111110 0 0
65 0 11111110 0 66 11111110 0 0

67 11111110 0 0
68 11111110 0 0
69 11111110 0 0
70 I 1 I 1 I 1 I 1 I 1 I 1 I 1 I 1 I 0 I 0

90
71 I 1 I 1 I 1 I 1 I 1 I 1 I 1 I 0 I 0 r 0
72 11111110 0 0
73 11111110 0 0
74 11111110 0 0
75 11111110 0 0
76 111111110 0
77 11111110 0 0
78 11111110 0 0
19 11111110 0 0
80 1111110 0 0 0
81 11111110 10
82 111111110 0
83 11111110 0 0
84 0000000000
85 0000000000
86 0000000000
87 0000000000
88 0000000000
89 0000000000
90 0000000000
91 0000000000
92 0000000100
93 0000000000
94 1000000000
95 0 1111110 0 0
96 0000000000
97 10 0 1110 0 0 0
98 111110 0 0 0 0
99 0000000000
100 0000000000
101 0 0 0 0 0 0 0 0 0 0
102 0000000000
103 0 110 0 10 10 0
104 0 11110 10 0 0
105 0 0 00 0 00000
106 OOOOOOOQOO
107 0 0 0 0 0 0 0 0 0 0
108 |o|o|o|o|o|o|o|o|o|o
The positive-transition number is then summed for the I, R and V applications and reported in TABLE 16:
TABLE 16:
c,^ . c • Transitions
Strains Species
ATFl ~C. albicans ~ 75 ATF2 C. albicans 75 ATF3 I C. albicans 75

91
ATF4 I C. albicans I 75
VIR31 C. albicans 61
ATF5 C. albicans IS
ATF6 C. albicans 75
ATF7 C. albicans 75
VIR32 C. (3/Z)/ca«5 68
VIR33 C. albicans 75
VIR34 C. albicans 11
VIR35 C. albicans IS
VIR36 C. albicans 73
VIR37 C a/Z)/cflW5 74
VIR38 C. a/j)/ca«5 75
VIR39 C. albicans IS
CA16 C. albicans 80
VIR43 £co// 12
VIR5 iS. aureus 2
VANS E..faecium 1
All the C albicans samples exhibit more than 60 positive transitions in the I category. All these samples are therefore confirmed as indeed belonging to the C. albicans species.
On the other hand, the VIR43, VIR5 and AST-VANS samples exhibit less than 13 positive transitions in the I category, these samples are therefore confirmed as not belonging to the C. albicans species.
The observation of transitions 84 and 85 indicates the presence of the mutated peptide
and reflects the resistance of the ATF6 and ATF7 strains.
The observation of transitions 86 and 87 indicates the presence of the mutated peptide
and reflects the resistance of the ATF2 and ATF3 strains.
The observation of transition 96 indicates the presence of the mutated peptide and
reflects the resistance of the ATF5 strain.
The observation of transitions 99 and 100 indicates the presence of the mutated peptide
and reflects the resistance of the ATF7 strain.
The observation of transitions 88 and 89 indicates the presence of the native peptide
and reflects the sensitivity of the ATF4 strain.
The observation of transitions 93 and 94 indicates the presence of the native peptide
and reflects the sensitivity of the ATFl and ATF4 strains.

92 All the areas of the I transitions are summed to give the sum SI. All the areas of the V transitions are summed to give the sum SV. The SV/SI ratio is then calculated and multiplied by a multiplication factor MF. The results obtained are given in TABLE 17. TABLE 17:
Sum (SI) of the ^u'^ (^V) of Ratioof the sums SV/SI ,,,,., ,.
r>x • r.u 1 the areas of ,,. , .. ^, ,^ ,. ^. Multiplication
Strains areas of the I .. ., multipled bythe multplication , , ,.,_,
, ... theV ft /,«CN factor (MF)
transitions . .,. factor (MF) ^ ''
transitions
ATF1 1.8E+07 1.6E+03 1.9E+03 2.0E+07
ATF2 1.3E+07 1.6E+03 2.4E+03
ATF3 2.0E+07 2.3E+03 2.3E+03
ATF4 2.9E+07 3.3E+03 2.3E+03
VIR31 1.6E+07 1.4E+06 1.8E+06
ATF5 1.7E+07 2.3E+03 2.6E+03
ATF6 2.4E+07 4.8E+03 4.0E+03
ATF7 2.9E+07 2.4E+03 1.7E+03
VIR32 2.9E+07 5.3E+05 3.6E+05
VIR33 1.9E+07 4.2E+04 4.4E+04
VIR34 4.2E+07 1.8E+03 8.9E+02
VIR35 1.1E+07 8.8E+03 1.5E+04
VIR36 8.3E+06 3.5E+04 8.5E+04
VIR37 2.3E+07 3.9E+03 3.4E+03
VIR38 2.4E+07 3.4E+03 2.8E+03
VIR39 1.6E+07 9.2E+03 1.1E+04
CA16 I 1.2E+07 I 5.4E+03 | 9.1E+03
The VIR31 and VIR32 strains, which have a (SV/SI) ratio x MF greater than 9x10'*, overexpress lipase 8, these strains are therefore virulent. All the other strains have a ratio less than 9x10 , they do not overexpress lipase 8 and are not therefore virulent. Interestingly, since the (SV/SI) ratio x MF is higher for the VIR31 strain than for the VIR32 strain, the VIR31 strain is characterized as more virulent than the VIR32 strain.
For the typing, the T-category transitions of each strain are compared with the transitions of the other strains considered as reference strains. In practice, a value 0 is assigned when the transitions between the two strains are classified in the same category (positive or negative) and a value of 1 is assigned when the transitions between the two strains are classified in different categories (a positive transition and a negative transition). The values are summed for all the T-category transitions of each strain pair in order to establish a score. The scores are given in TABLE 18:

93 TABLE 18:
StrainATF1ATF2ATF3ATF4ATF5ATF6ATF7VIR31VIR32VlR33VIR34VIR35VIR36VlR37VIR38VIR39CA16VIR43VlR5^^^g
ATF1 0254438 39 30 23 23 25 25 24 23 22 16 70 78 79
ATF2 2 ~0~ 3 6 6 3 10 41 32 25 25 25 25 26 25 24 16 70 78 79
ATF3 5 3 0 9 9 6 9 40 31 26 24 24 24 25 24 23 15 69 77 78
ATF4 4 ~6 9~_0 8 7 12 41 32 25 27 27 27 26 25 26 18 72 82 83
ATF5 4 6 9 8 0 7 10 37 28 21 25 23 23 22 21 20 14 70 78 79
ATF6 3 3 6 7 7 0 7 42 33 26 26 26 26 27 26 25 17 71 79 80
ATF7 8 10 9 12 10 7 0 41 32 25 23 25 25 24 23 24 18 72 80 81
VIR31 39 41 40 41 37 42 41 0 9 22 26 22 20 21 22 21 29 65 69 70
VIR32 30 32 31 32 28 33 32 9 0 13 19 13 13 12 13 14 22 66 74 75
VIR33 23 25 26 25 21 26 25 22 13 0 8 4 6 5 4 5 15 71 79 80
VIR34 23 25 24 27 25 26 23 26 19 8 0 10 12 9 8 9 19 71 73 74
VIR35 25 25 24 27 23 26 25 22 13 4 10 0 2 3 4 5 11 69 77 78
VIR36 25 25 24 27 23 26 25 20 13 6 12 2 0 5 6 7 13 67 75 76
VIR37 24 26 25 26 22 27 24 21 12 5 9 3 5 0 1 6 12 70 76 77
VIR38 23 25 24 25 21 26 23 22 13 4 8 4 6 1 0 5 13 71 77 78
VIR39 22 24 23 26 20 25 24 21 14 5 9 5 7 6 5 0 14 68 76 77
CA16 16 16 15 18 14 17 18 29 22 15 19 11 13 12 13 14 0 72 80 81
VIR43 70 70 69 72 70 71 72 65 66 71 71 69 67 70 71 68 72 ~ ~ ~
VIR5 78 78 77 82 78 79 80 69 74 79 73 77 75 76 77 76 80 ~ ~ ~
A<;T-
VAN8 ^^ ^® ^^ ^^ ^^ ^° ^^ ^° '^^ ^° ^'* ''^ ^^ ^^ 78 77 81 ~ ~ ~
The strains which have a score less than or equal to 2 are of the same type, the strains which have a score strictly greater than 2 are of different type. Thus, the ATFl and ATF2, VIR35 and VIR36, and VIR37 and VIR38 strains are respectively of the same type. All the other strains taken in pairs are of different types. The high sums obtained between the VIR43, VIR5 and AST-VANS strains, which are not S. aureus, and all the other strains, which are S. aureus, should be noted. These results confirm the specificity of the method.
The VIR43, VIR5 and AST-VANS strains are of different species. These strains cannot therefore be compared with one another and no value is reported in TABLE 18. Extremely advantageously, scores greater than 20, for instance between ATFl and VIR39, reflect a great divergence between strains. Scores between 14 and 20, as between CA16 and ATFl, reflect a moderate divergence and scores between 4 and 14, as between ATFl and ATF7, a weak divergence.

94 The method thus implemented therefore makes it possible not only to establish whether two strains are of the same type, which is important for identifying a common seat of infection, but also to estimate the proximity of two strains, which is extremely important for epidemiological studies.
This example shows that, very advantageously, the present invention makes it possible, in a time of less than one hour, which is very short, to confirm the identity of a species such as C. albicans and to determine, simultaneously within the same analysis, the properties of typing and potential resistance to at least one antibiotic and to establish the existence of a virulence factor. The present invention also allows quantitative assaying, which is particularly advantageous when the properties of resistance to at least one antibiotic, or virulence, are linked to the level of expression of a protein or of a metabolite. This is what we illustrate here with the quantitative assaying of lipase 8. These properties were established with the same instrument, which greatly facilitates the analysis and the reporting of the results. Finally, the characteristics of yeasts are established using fungal proteins, which reflects the existence of live and viable microorganisms, unlike characterizations using fungal DNA which can be distorted by the existence of dead yeasts.
Example 10: Protocol for digestion of microorganisms in the presence of methanol, suitable for assaying at least one metabolite
Conventionally, the following protocol is implemented in 19 steps:
1. Sampling of five microorganism colonies, obtained according to example 1, and suspension in 100 |^1 of a 6M guanidine hydrochloride, 50 mM Tris-HCl solution, pH=8.0.
2. Centrifligation at 15000g for 5 minutes.
3. Pellet taken up in 400 |al of a 50 mM ammonium bicarbonate solution, pH=8.0.
4. Addition of 600 \i\ of methanol.
Steps 5 to 9: idem steps 2 to 6 of example 4. Steps 10 to 19: idem steps 8 to 17 of example 6.

95 Example 11: Characterization of C albicans samples by simultaneous analysis of proteins and of metabolites;
After having established the one or more species of the samples according to any one of the methods described in examples 1 to 3, the species listed below are analyzed.
Five strains of C. albicans are analyzed in order to confirm their identification and to
establish their characteristics:
ATFl ATF2
ATF3 ATF4
ATF6
Each sample is treated according to example 10, then a volume of 20 |J.l of digested proteins is injected and analyzed according to the same conditions as in example 7. Tables 13 and 14 are identical, with one exception. The transition number 109 is added to the method. It corresponds to the molecule, ergosterol, with clinical interest I and R. The charge state of the precursor is 1, the retention time is 36.3 minutes, the m/z filtered in Ql is 379.4, the m/z filtered in Q3 is 69.2, and the collision energy is 47. The mass parameters are identical to those of example 7, with the exception of those below:
Curtain gas: 25.00 psi
Source temperature: 450.00°C
Heating gas: 50.00 psi
Entry potential before QO (EP): 4.00 V
For transition 109, parameters differ:
Declustering potential (DP): 200.00 V
Entry potential before QO (EP): 10.00 V
Collision cell exit potential (CXP): 8 V
All the transitions 1 to 108 are analyzed in a maimer identical to example 9. All the areas of the I transitions are summed to give the sum SI. The area of transition 109 is called A109. The A109/SI ratio is then calculated and multiplied by a multiplication factor MF. The results obtained are given in table 19.

96 TABLE 19:
I I ] \2 ,o,, ,. I A109/SI ratio I
Area of Sum (SI) of the ,.. ,. . . „ ,, ,,.,.,. ,.
„^ . ^ ,.■ Af^r, to, , multiplied by the Multiplification
Strams transition 109 areas of the I u- i- i t T /n/iir(A109) transitions TT':!!^!' f''*°'('^'^)
^ ' factor(MF)
ATF1 1.7E+03 9.3E+07 1.6E+03 9.0E+07
ATF2 7.9E+03 1.1E+08 6.4E+03
ATF3 3.9E+03 5.7E+07 6.3E+03
ATF4 5.7E+03 2.9E+08 1.8E+03
ATF6 I 6.4E+03 | 6.8E+07 | 8,5E+03
The ATF2, ATF3 and ATF6 strains, which have a (A109/SI) ratio x MF greater than 2x10 , overexpress ergosterol; these strains are therefore resistant. All the other strains have a ratio less than 2x10 , they do not express ergosterol and are therefore not resistant.
This example shows that, very advantageously, the present invention makes it possible to simultaneously assay, within the same analysis, in a time of less than one hour, which is very short, and quantitatively, various compounds such as proteins, peptides or metabolites. Even more advantageously than the assaying of a protein, the quantitative assaying of a metabolite resulting from the action of this protein characterizes the presence of a functional protein and more broadly of a functional synthesis pathway. Thus, the quantification of ergosterol makes it possible in this case to establish the existence of a mechanism of resistance to fluconazole linked to the existence of functional and strongly active lanosterol demethylase.
Example 12: Characterization of microorganisms present in urine samples:
After having established the one or more species of the samples according to any one of the methods described in examples 1 to 3, five urine samples contaminated with E. coli (Urine 1 to 5) are analyzed. A noncontaminated sixth urine (Urine 6) is also analyzed in order to serve as a negative control.
The following protocol is implemented in 29 steps (steps 5 to 12 are optional and could be omitted without significantly altering the results):
1. Centriftagation of 5 ml of contaminated urine at 2000g for 3 0 seconds.
2. Recovery of the supernatant.
3. Centrifugation at 15000g for 5 minutes.

97
4. Removal of the supernatant.
5. Washing of the pellet with 3 ml of distilled water by resuspension.
6. Centrifugation at ISOOOg for 5 minutes.
7. Removal of the supernatant.
8. Bringing the pellet into contact with solvent in a dilution to 1/10.
9. Leaving for one hour at -20°C.
10. Centrifugation at ISOOOg for 5 minutes.
11. Removal of the supernatant.
12. Bringing the pellet into contact with solvent in a dilution to 1/10.
13. Leaving for one hour at -20°C.
14. Centrifugation at ISOOOg for 5 minutes.
15. Removal of the supernatant.
12. Suspension of the pellet in 10 to 100 |al of a 6M guanidine hydrochloride,
SO mM Tris-HCl solution, pH=8.0.
13. Steps 14 to 18: idem steps 2 to 6 of example 4.
19. Steps 19 to 28: idem steps 8 to 17 of example 6.
29. Injection of 100 ^\ of acidified eluate onto the chromatographic system and mass spectrometer, according to the protocol described in example 7.
The areas obtained for each of the transitions and for each of the microorganisms studied were measured. Since the urine samples have a bacterial load that is very different from one sample to another, the threshold used in example 7 for declaring a positive transition cannot be used here. It must be adjusted to the amount of bacteria present. In the previous example, the sampling of a colony resulted in a comparable amount of microorganisms. This is no longer the case in this example. Thus, the amount of bacteria is estimated by summing the area of all the I transitions. The sum of the areas of the I transitions is reported in TABLE 20.
TABLE 20:
Urine 1 Urine 2 Urine 3 Urine 4 Urine 5 Urine 6
1509 251.3 1 828 079.5 | 5 349 271.0 | 11 918 946.9 | 1 054 480.6 | 0.0

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Thus, sample 4 comprises a bacterial load that is higher than sample 3, which itself has a higher load than examples 1 and 5, which themselves have higher loads than sample 2. Samples 1 and 5 have a comparable bacterial load. Sample 6 does not comprise an E. coli and exhibits a zero load, thereby demonstrating the specificity of the technique.
The area of each transition is then standardized by dividing it by the area of all the I transitions. All the transitions of which the ratio is greater than or equal to 0.00015 (non-dimensional ratio) are considered to be positive and have been denoted "1" in TABLE 21. All the transitions of which the ratio is less than 0.00015 are considered to be negative and have been denoted 0 in TABLE 21. When no signal peak was observed, the transition was noted as negative.
TABLE 21:
, Urine 1 Urine 2 Urine 3 Urine 4 Urine 5 Urine 6
number
1 1 11110
2 1 11110
3 \ 11110
4 0 0 1110
5 0 0 1 ' 1 1 0
6 0 0 0 110
7 0 0 0 0 0 0
8 0 0 0 0 0 0
9 0 0 0 0 0 0
10 1 11110
11 1 11110
12 1 11110
13 1 11110
14 1 11110
15 1 11110
16 1 11110
17 1 11110
18 1 1110 0
19 1 1110 0
20 1 0 10 0 0
21 1 11110
22 1 11110
23 1 11110
24 \ 11110
25 1 10 110
26 1 10 110
27 \ 10 110
" 28 I 1 I 1 I 1 I 1 I 1 I 0

99
29 I 1 I 1 I 1 I 1 I 1 I 0
30 1 11110
31 0 11110
32 0 0 1110
33 0 0 0 10 0
34 1 11110
35 1 11110
36 \ 11110
37 1 11110
38 I 11110
39 I 11110
40 1 11110
41 1 11110
42 1 11110
43 1 11110
44 1 11110
45 1 11110
46 0 0 1110
47 0 0 1110
48 0 0 1110
49 0 0 0 0 0 0
50 0 0 0 0 0 0
51 0 0 0 0 0 0
52 0 0 0 0 0 0
53 0 0 0 0 0 0
54 0 0 0 0 0 0
55 1 110 10
56 1 110 10
57 11 1 0 1 0
58 1 110 10
59 1 1 1 0 10
60 1 110 0 0
61 0 0 0 0 0 0
62 0 0 0 0 0 0
63 0 0 0 0 0 0
64 0 0 0 0 0 0
65 0 0 0 0 0 0
66 1 0 0 0 0 0
67 1 11110
68 1 11110
69 1 11110
70 1 0 0 110
71 1 0 0 110
72 0 0 0 0 0 0
73 1 0 1 1 10
74 1 0 1 1 10
75 1 0 1110
76 1 11110
77 1 11110
78 I 1 I 1 I 1 I 1 I 1 I 0

100
79 I 1 I 1 I 1 I 1 I 1 I 0
80 1 11110
81 1 11110
82 1 11110
83 1 11110
84 1 11110
85 1 11110
86 1 11110
87 1 11110
88 1 11110
89 1 11110
90 1 11110
91 \ 0 1110
92 1 11110
93 1 0 1110
94 0 0 0 0 0 0
95 0 0 0 0 0 0
96 0 0 0 0 0 0
97 0 0 0 0 0 0
98 0 0 0 0 0 0
99 0 0 0 0 0 0
100 0 0 0 0 0 0
101 0 0 0 0 0 0
102 0 10 0 0 0
103 1 11110
104 1 11110
105 1 11110
106 1 11110
107 1 11110
108 1 11110
109 1 11110
110 1 11110
111 1 11110
112 1 11110
113 1 11110
114 0 0 0 110
115 1 11110
116 1 11110
117 1 11110
118 0 0 0 0 0 0
119 0 0 0 0 0 0
120 0 0 0 0 0 0
121 1 110 0 0
122 0 0 0 0 0 0
123 0 0 0 0 0 0
124 0 0 0 0 0 0
" 125 I 0 I 0 I 0 I 0 I 0 I 0

101 The positive-transition number is then summed for the I, R and V applications and reported in TABLE 22:
TABLE 22:
Urine 1 Urine 2 Urine 3 Urine 4 Urine 5 Urine 6
I 36 36 40 44 41 0
R 6 6 6 0 5 0
V |i|i|i|o|o|o
In the same way as the transition positivity threshold had to be modified in order to take into account the concentration of the samples, the number of peptides necessary for characterizing a strain must be adjusted to the total concentration of bacteria. Certain weakly detected peptides in example 7 may be under the limit of detection if the amount of bacteria is less than one colony.
Urines 1 to 5 exhibit more than 30 positive transitions in the I category. All these samples are therefore confirmed as contaminated with the E. coli species.
On the other hand, urine 6 exhibits no positive transition in the I category. It is therefore confirmed as not being contaminated with the E. coli species.
Urines 1, 2, 3 and 5 exhibit at least five positive transitions for the R category, they therefore express the plasmid-mediated penicillinase TEM-2, which is synonymous with a mechanism of resistance to penicillins, in particular aminopenicillins or A penicillins (ampicillin), carboxypenicillins or C penicillins (ticarcillin) and ureidopenicillin or penicillin U (piperacillin).
On the other hand, urines 4 and 6 do not exhibit positive transitions for the R category, they do not therefore express the plasmid-mediated penicillinase TEM-2. These strains are therefore sensitive to penicillins.
These results of resistance to ampicillin, ticarcillin and piperacillin were confirmed with the VITEK 2 automated device sold by the applicant and the AST-EXN and AST-N103 cards. The confirmation took a time of 6 to 8 hours on the V1TEK®2 automated device, which is slower than for the present invention.

102 Urines 1 to 6 do not exhibit more than one positive transition for the V category. These urines are not therefore contaminated with toxins of shigatoxin type.
For the typing, the T-category transitions of each strain are compared with the transitions of the other strains considered as reference strains. In practice, a value 0 is assigned when the transitions between the two strains are classified in the same category (positive or negative) and a value of 1 is assigned when the transitions between the two strains are classified in different categories (a positive transition and a negative transition). The values are summed for all the T-category transitions of each strain pair in order to establish a score. The scores are given in TABLE 23:
TABLE 23:
Urine 1 Urine 2 Urine 3 Urine 4 Urine 5
Urinal 0 ~~[]~ 13 ~ 19 15
Urine 2 11 0 16 24 20
Urine 3 13 16 0 16 12
Urine 4 19 24 16 0 8
Urine sl 15 | 20 | 12 | 8 | 0
No urine has a score less than or equal to 4. The strains infecting urines 1 to 5 are therefore of different types. These urine samples were collected in a local medical test laboratory where infections often occur in different environments. Under these conditions, it is uncommon to observe urine infections linked to the same strain.
This example shows that, very advantageously, the present invention makes it possible, directly from the primary sample, which is very advantageous, to confirm the identity of a species such as E. coli and to determine, simultaneously within the same analysis, the properties of typing and potential resistance to at least one antibiotic.

103 Literature references
[I] J. Anhalt & C. Fenselau, 1975, Anal. Chem., 47(2):219-225.
[2] A. Fox et al., ed., 1990, Analytical microbiology methods: chromatography and
mass spectrometry, Plenum Press, New York, N.Y.
[3] M. Claydon et al, 1996, Nature Biotech. 14:1584-1586.
[4] T. Krishnamurthy & P. Ross, 1996, Rapid Com. Mass Spec, 10:1992-1996.
[5] P. Seng et al. 2009, Clin. Infect. Dis., 49:543-551.
[6] C. Fenselau et al, 2008, Appl. Environ. Microbiol., 904-906.
[7] D. Ding et al. 2009, J. Pharm. Biomed. Anal. 50:79-85.
[8] R. Everley et al, 2009, J. Microbiol. Methods, 77:152-158.
[9] S. Hofstadler et al., 2005, Int. J. Mass Spectrom., 242:23-41.
[10] D. Ecker, 2008, Nat. Rev. Microbiol, 6(7):553-558.
[II] W.-J. Chen et al, 2008, Anal. Chem., 80: 9612-9621.
[12] D. Lopez-Ferrer et al, 2008, Anal. Chem., 80:8930-8936.
[13] D. Lopez-Ferrer et al., 2005, J. Proteome res., 4(5): 1569-1574.
[14] T. Fortin et al, 2009, Mol. Cell Proteomics, 8(5): 1006-1015.
[15] H. Keshishian et al., 2007, Mol. Cell Proteomics, 2212-2229.
[16] J. Stal-Zeng et al., 2007, Mol. Cell Proteomics, 1809-1817.
[17] Gaskell, Electrospray: principles and practise, 1997, J. Mass Spectrom., 32, 677-
688).
[18] V. Fusaro et al, 2009, Nature Biotech. 27,190-198.
[19] J. Mead et al., 15 Nov 2008, Mol. Cell Proteomics, E-pub.
[20] F. Desiere et al., 2006, Nucleic Acids Res., 34(database issue): D655-8.
[21] L. Anderson & C. Hunter, 2006, Mol. Cell Proteomics, 573-588.
[22] B. Han & R. Higgs, 2008, Brief Funct Genomic Proteomic.,7(5):340-54.
[23] K.-Y. Wang et al., 2008, Anal Chem, 80(16) 6159-6167.
[24] J. Bundy & C. Fenselau, 1999, Anal. Chem. 71: 1460-1463.
[25] K-C Ho et al., 2004, Anal. Chem. 76: 7162-7268.
[26] Y.S. Lin et al., 2005, Anal. Chem., 77: 1753-1760.
[27] S. Vaidyanathan et al., 2001, Anal. Chem., 73:4134-4144.
[28] P. Seng et al., 2009, Clin. Infect. Dis., 49:543-551.
[29] Manes N. et al., 2007, Mol. & Cell. Proteomics, 6(4): 717-727.

104 [30] R. Nandakumar et al., 2009, Oral Microbiology Immunology, 24:347-352. [31] L. Hemychova et al., 2008, Anal. Chem., 80:7097-7104. [32] J.-M. Pratt et al., 2006, Nat. Protoc, 1:1029-1043. [33] V. Brun et al, 2007, Mol. Cell Proteomics, 2139-2149.

105 CLAIMS
1. A method for characterizing at least one microorganism from a sample, which comprises identifying said at least one microorganism and determining the properties of typing, potential resistance to at least one antimicrobial, and virulence factor, characterized in that the determining of the properties of typing, resistance to at least one antimicrobial, and virulence factor for said at least one microorganism is implemented by mass spectrometry using proteins, peptides and/or metabolites as markers of said properties of typing, resistance to at least one antimicrobial, and virulence factor.
2. The method for characterizing at least one microorganism as claimed in claim
1, characterized in that the mass spectrometry, implemented for the properties of
typing, potential resistance to at least one antimicrobial, and virulence factor, is
spectrometry of MS/MS type.
3. The method for characterizing at least one microorganism as claimed in claim
2, characterized in that the MS/MS spectrometry is MRM.
4. The method for characterizing at least one microorganism as claimed in any one of claims 1 a 3, characterized in that the determining of the properties of typing, resistance to at least one antimicrobial, and virulence factor is implemented in the same mass spectrometry apparatus.
5. The method for characterizing at least one microorganism as claimed in claim 4, characterized in that the determining of the properties of typing, resistance to at least one antimicrobial, and virulence factor is implemented simultaneously.
6. The method for characterizing at least one microorganism as claimed in any one of the preceding claims, characterized in that the identifying of said at least one microorganism is also implemented by mass spectrometry.

106
7. The method for characterizing at least one microorganism as claimed in claim 6, characterized in that the mass spectrometry for identifying said at least one microorganism is spectrometry of MS type, of MS/MS type or of the type MS followed by spectrometry of MS/MS type.
8. The method for characterizing at least one microorganism as claimed in any one of the preceding claims, characterized in that it comprises an additional step of confirming the identification of said at least one microorganism, which confirmation step is implemented by mass spectrometry.
9. The method for characterizing at least one microorganism as claimed in claim 8, characterized in that the mass spectrometry of the confirmation step is mass spectrometry of MS/MS type.
10. The method for characterizing at least one microorganism as claimed in
claim 9, characterized in that the mass spectrometry of the confirmation step is an
MRM.
11. The method for characterizing at least one microorganism as claimed in any one of the preceding claims, characterized in that the markers of said properties of typing, resistance to at least one antimicrobial, and virulence factor are proteins of said at least one microorganism.
12. The method for characterizing at least one microorganism as claimed in claim 11, characterized in that the proteins of said microorganism are digested into peptides.
13. The method for characterizing at least one microorganism as claimed in claim 12, characterized in that the digestion is carried out with an enzyme.
14. The method for characterizing at least one microorganism as claimed in claim 13, characterized in that the enzyme is trypsin.

107
15. The method for characterizing at least one microorganism as claimed in the preceding claims, characterized in that said at least one microorganism is a bacterium and the antimicrobial is an antibiotic.
16. The method for characterizing at least one microorganism as claimed in claim 15, characterized in that the bacterium is Staphylococcus aureus.
17. The method for characterizing at least one microorganism as claimed in claim 16, characterized in that the determining of the typing properties uses at least one peptide having the sequence SEQ ID No.2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17,19, 20, 21, 23,24,26, 27, 29, 30, 32, 34, 35, 37, 38,40,42,44 or 45.
18. The method for characterizing at least one microorganism as claimed in claim 16 or 17, characterized in that the determining of the properties of potential resistance to at least one antibiotic uses at least one peptide having the sequence SEQ IDNo.l0tol7.
19. The method for characterizing at least one microorganism as claimed in toy one of claims 16 to 18, characterized in that the determining of the virulence uses at least one peptide having the sequence SEQ ID No. 19, 20, 21, 23 or 24.
20. The method for characterizing at least one microorganism as claimed in any one of claims 16 to 19, characterized in that the identifying uses at least one peptide having the sequence SEQ ID No.26, 27, 29, 30, 32, 34, 35, 37, 38, 40, 42, 44 and 45.
21. The method for characterizing at least one microorganism as claimed in claim 15, characterized in that the bacterium is Escherichia coli.
22. The method for characterizing at least one microorganism as claimed in claim 21, characterized in that the determining of the typing properties uses at least one peptide having the sequence SEQ ID No.46 to 87.

108
23. The method for characterizing at least one microorganism as claimed in claim 21 or 22, characterized in that the determining of the properties of potential resistance to at least one antibiotic uses at least one peptide having the sequence SEQ ID No.85, 86 and 87.
24. The method for characterizing at least one microorganism as claimed in any one of claims 21 to 23, characterized in that the determining of the virulence uses at least one peptide having the sequence SEQ ID No. 62 to 66.
25. The method for characterizing at least one microorganism as claimed in any one of claims 21 to 24, characterized in that the identifying uses at least one peptide having the sequence SEQ ID No.46 to 61.
26. The method for characterizing at least one microorganism as claimed in claims 1 to 14, characterized in that said at least one microorganism is a yeast and the antimicrobial is an antifungal.
27. The method for characterizing at least one microorganism as claimed in claim 26, characterized in that the yeast is Candida albicans.
28. The method for characterizing at least one microorganism as claimed in claim 27, characterized in that the determining of the typing properties uses at least one peptide having the sequence SEQ ID No.88 to 125 and 177.
29. The method for characterizing at least one microorganism as claimed in claim 27 or 28, characterized in that the determining of the properties of potential resistance to at least one antifungal uses at least one peptide having the sequence SEQ ID No. 117 to 122 and 177.
30. The method for characterizing at least one microorganism as claimed in any one of claims 27 to 29, characterized in that the determining of the virulence uses at least one peptide having the sequence SEQ ID No. 123 to 125.

109
31. The method for characterizing at least one microorganism as claimed in any one of claims 27 to 30, characterized in that the identifying uses at least one peptide having the sequence SEQ ID No.88 to 116.
32. The method for characterizing at least one microorganism as claimed in any one of the preceding claims, characterized in that the metabolite is ergosterol.
33. A peptide having the sequence SEQ ID No.2, 3, 4, 5, 6, 7, 8, 10, 11, 12, 13, 14, 15, 16, 17, 19, 20, 21, 23, 24, 26, 27, 29, 30, 32, 34, 35, 37, 38, 40, 42, 44 to 125 and 177.
Dated this 08/05/2012 [ LA /I
[HRISHI^HI^Y CHMTOHURY]
V OFREMFRY& SAGAR
ATTORNEY FOR THE APPlicANT[S]