Identification of microorganisms based on peptide identification using a liquid separation device coupled to a mass spectrometer and processing means.

Abstract

The present invention relates to a method for identifying at least one microorganism present in a sample based on the detection of peptides resulting from the cleavage of a ribosomal protein of said microorganism, comprising steps a), b), c), d), e), f), g), h) and i) as described herein, wherein each transition read from a predefined list is associated with a peptide and the microorganism is identified according to the detection of said peptides.

Description

[Technical field]

[0001] The present invention relates to a method for identifying microorganisms in a biological sample, in particular from a blood sample of human origin, which method can be used to detect sepsis, i.e. a blood-borne infection. [Background technology]

[0002] Identifying the microorganism causing an infection is an essential step in managing a patient. Rapid access to this identification information is even more important in the case of symptomatic bacteremia, which is caused by the persistent spread of pathogens from the site of infection and can subsequently lead to the septic syndrome. Sepsis can range from mild to severe and eventually develop into septic shock, with a mortality rate of up to 80%.

[0003] Therefore, rapid identification of the pathogen causing bacteremia is an important step to be able to direct the choice of antibiotic therapy or to proceed with tapering of the initial broad-spectrum antibiotic therapy. The hope of such a rapid diagnosis is not only to reduce the side effects of treatment in patients but also to limit the contribution of antibiotic use to the emergence of new antibiotic-resistant strains or resistance mechanisms.

[0004] In areas outside of human health, the rapid identification of microorganisms present in a sample is also a key element for adapting antimicrobial strategies.

[0005] In the past two decades, two techniques have emerged that have revolutionized microbial identification by significantly shortening the identification time compared to previously used biochemical techniques: Maldi-Tof mass spectrometry and molecular biology-based tools.

[0006] The principle of pathogen identification by matrix-assisted laser desorption / ionization-time of flight (Maldi-Tof) mass spectrometry is based on the comparison of experimental mass spectra of fingerprints of low molecular weight proteins released by lysis of the microorganism of interest with a database containing tens of thousands of mass spectra of fingerprints obtained from known microbial strains. A match score is then established to identify the pathogen genus or the pathogen genus and species. The principle of microbial identification by Maldi-Tof was first described in 1999 for low molecular weight proteins (Holland et al., 1999).

[0007] This technology was subsequently widely adopted in hospitals under the brands VITE® MS (Biomerieux) and MALDI Biotyper® (Brucker Daltonics).

[0008] When applied to blood samples, blood cells need to be lysed and removed to limit sources of interfering signals on the mass spectrum. Patent US8569010 discloses a protocol based on the use of sodium dodecyl detergent to aid in efficient blood cell lysis prior to Maldi-Tof analysis.

[0009] Compared to biochemical identification techniques that require prior isolation of microorganisms, Maldi-Tof technology can reduce the identification time by approximately 24 hours and therefore have a direct impact on mortality statistics or average length of stay. Furthermore, the calculated analytical cost per sample is very low and the technology produces almost no hospital waste. These favorable health economic characteristics explain the rapid introduction of Maldi-Tof into hospitals.

[0010] However, the Maldi-Tof method has limitations. For example, it is difficult to identify phylogenetically close species (i.e., Escherichia coli ( Escherichia coli ) / Shigella spp.( Shigella ), Citrobacter freundii ( Citrobacter freundiiIdentification of bacteria belonging to the Enterobacter cloacae group (e.g., members of the Enterobacter cloacae group) is difficult. Similarly, in polymicrobial infections, identification is difficult due to overlapping fingerprints or poor expression of one organism compared to another predominant species. Such situations are commonly encountered in the context of bacteremia related to polymicrobial infections of the gastrointestinal tract (peritonitis, intraperitoneal abscesses).

[0011] This low sensitivity is less of an issue for techniques based on molecular biology. Commercially available solutions using molecular biology in the broad sense can be distinguished into four categories, depending on whether they employ methods based on i) fluorescent in-situ hybridization (FISH), ii) DNA microarray hybridization, iii) nucleic acid amplification (PCR), or iv) a combination of them.

[0012] An ideal pathogen identification technology should cover most species associated with sepsis, be transferable directly from positive blood culture aliquots, be economically viable, allow identification of the different pathogens constituting a polymicrobial infection in the shortest possible time (ideally less than 1 h), and concomitantly allow estimation of relative or absolute quantification of pathogens. Ideally, this technology should be implemented in a single analytical platform and somehow be able to simultaneously or sequentially identify antibiotic resistance mechanisms or susceptibility profiles that may be associated with the identified pathogens.

[0013] Recently, the potential interest of using liquid chromatography-mass spectrometry in conjunction with bottom-up proteomic analytical approaches to identify bacteria has been evaluated in several exploratory studies. In this approach, the protein content of bacteria or yeast is subjected to specific enzymatic digestion to generate peptides, which are then partially separated by a chromatographic step and reconstituted into characteristic ions to generate mass spectra and / or chromatograms, which are compared to public or proprietary databases. Mass spectrometry can be performed in a non-targeted or targeted manner.

[0014] In the case of non-targeted analysis, the mass spectrometer can be operated in such a way that it obtains information about the exact (monoisotopic) mass or chemical mass or molecular mass or average mass of each of the peptides in the mixture obtained, for example, from the enzymatic hydrolysis. In this case, a simple analysis called MS or MS1 is performed, in which, as in Maldi-Tof, the experimental fingerprints (or mass-to-charge ratio values; m / z) of all the masses of the peptides obtained from the enzymatic digestion are compared with all the theoretical fingerprints obtained by the same enzymatic digestion of the entire bacterial or yeast proteome. This is an approach called LC-MS1 as described in (Lasch et al. 2020).

[0015] In another embodiment, the process includes a step of fragmenting peptides in addition to or instead of peptide mass information. This step can be conditioned by a preliminary observation at time t of a chromatogram of n masses (or mass-to-charge ratio values; m / z) of intact peptides. This mode of operation is called Data Dependent Acquisition (DDA), also known as shotgun proteomics or Information Dependent Acquisition (IDA). The experimental fragmentation spectra of peptides, combined with or without information on their masses, are then compared with theoretical fragmentation spectra of all peptides obtained from enzymatic digestion of bacterial or yeast proteomes to identify pathogens. This process is used, for example, in (Boulund et al. 2017).

[0016] Alternatively, peptides are not individually selected from the mass spectrum but are systematically fragmented in a blinded fashion following an acquisition format called Data Independent Acquisition (DIA), also known as Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH) or MSE, a method used by (Blumenscheit et al., 2020) to detect peptides arising from enzymatic digestion of proteins involved in antibiotic resistance.

[0017] The targeted acquisition modes are called Selected Reaction Monitoring (SRM), Multiple Reaction Monitoring (MRM), Parallel Reaction Monitoring (PRM), Multiple Reaction Monitoring-High Resolution (MRM-HR), and Multiple Reaction Monitoring cubed (MRM3). Several studies have been reported on the implementation of targeted mass spectrometry for the identification of bacteria or yeasts, for example in urine, tracheobronchial aspirates, or from isolated colonies. This method has also been implemented for typing bacteria of the genus Acinetobacter, for the detection and quantification of toxins, and for the detection of antibiotic resistance mechanisms.

[0018] International application WO2011 / 045544 describes a method for the detection of Staphylococcus aureus (Staphylococcus aureus) bacteria from isolated colonies ( Staphylococcus aureus ) describe the use of this targeted mass spectrometry method in combination with a chromatographic separation system for strain typing and the parallel detection of virulence factors and antibiotic resistance. Similarly, applications WO2012 / 143535 and WO2012 / 143534 describe the use of this same method for detecting proteins associated with various antibiotic resistance mechanisms.

[0019] However, it should be noted that so far there is no method for diagnosing bloodstream infections based on this method of combining peptide isolation and their detection by mass spectrometry, for two reasons.

[0020] Firstly, these analytical methods are too time consuming, in most cases chromatographic separations lasting 30-120 min, which limits the number of samples that can be analyzed per day and increases the analytical costs.

[0021] Secondly, in targeted mass spectrometry, when a large number of targets are sampled, the signals of these targets must be tracked only in the chromatographic retention time window in which they are expected to be detected. This requires measuring the signal intensity of the compounds eluted from the chromatographic separation system at least eight times in order to be able to define the shape of the chromatographic peak of the compound with sufficient accuracy. This approach is called "scheduled MRM, scheduled MRM HR, timed MRM, dynamic MRM" by mass spectrometry manufacturers. The drawback is that if the retention time changes unexpectedly (e.g. due to the effects of sample composition or concentration, or wear of the chromatographic column), the target compound may fall outside the scheduled retention window and go undetected. Taking this limitation into account, users usually take a sufficiently wide retention time window, but this precaution implies a reduction in the number of compounds that can be detected by this method.

[0022] Patent EP3384517 describes a technique that overcomes these limitations. The method relies on the monitoring of "sentinel signals" belonging to compounds spread over the chromatographic separation scale. When a sentinel signal is detected above a defined threshold, it triggers the monitoring of a set of signals specific for the target molecule of interest until a new sentinel signal is detected. Thus, all target compounds continue to be reliably detected despite drifts in retention times. Summary of the Invention

[0023] The present patent application describes a method for rapid identification of microorganisms in less than 10 minutes, preferably 5-7 minutes, targeting biomarker peptides selected exclusively from peptides derived from enzymatic digestion of ribosomal proteins of said microorganisms.

[0024] The method is based on a list of thoroughly selected peptides to carry out this identification method, which are specific to the species to be identified and which possess physicochemical properties that allow them to meet the specifications set for the separation step, i.e. to have an optimal peak capacity during a gradient time of about 5 minutes.

[0025] The present invention relates to a method for identifying at least one microorganism present in a sample, based on the detection of peptides resulting from cleavage of a ribosomal protein of said microorganism, comprising the following steps: a) lysing microorganisms and cleaving proteins present in said sample to obtain a peptide mixture; b) decomplexing the peptide mixture using a liquid separation device connected to a mass spectrometer; c) atomizing the liquid eluted from the separation device using an ion source to generate an ion current; d) receiving, with the mass spectrometer, the ion current from the ion source and performing, for each cycle of a plurality of cycles, a series of filtering steps on the ion current to detect transitions, the transitions including a precursor ion and at least one fragment ion of the precursor ion, the transitions being read from a predefined transition list using the mass spectrometer, and for each transition in the series of transitions, the mass spectrometer selecting and fragmenting a precursor ion of each transition; e) receiving, with a processor, data regarding a plurality of transitions used to monitor the peptide mixture; f) using a processor, allocating the plurality of transitions into two or more consecutive transition groups and assigning them to the predefined transition list; g) monitoring, with a processor, at least one sentinel transition associated with a sentinel compound in each of two or more consecutive groups, wherein the at least one sentinel transition is selected as having the slowest predicted retention time in the group; h) when a signal of at least one sentinel transition of a group is detected by the mass spectrometer, using the processor to stop monitoring the transitions of the previous group while starting monitoring at least one transition of the next successive group; i) optionally using a processor to generate a chromatogram or electropherogram from the detection of transitions read from a predefined list by said mass spectrometer; wherein each transition read from the predefined list is associated with a peptide, and a microorganism is identified according to the detection of said peptide.

[0026] The present invention also relates to a system for carrying out the method as defined above, comprising a mass spectrometer connected to a liquid separation device and adapted to carry out steps e), f), g) and h), in particular receiving data regarding a plurality of transitions used to monitor a peptide mixture; Assigning a plurality of transitions into two or more consecutive transition groups and assigning them to the predefined transition list; monitoring at least one sentinel transition in each of said two or more consecutive groups; when a signal of at least one sentinel transition of a group is detected by the mass spectrometer, starting monitoring of at least one sentinel transition of a next successive group; and In some cases, generate chromatograms or electropherograms and a processing means adapted to said system.

[0027] The present invention also relates to a group of peptides suitable for carrying out the method as described above, said peptides originating from ribosomal proteins, containing between 6 and 20 amino acids and being decomplexed in the decomplexing step using a mobile phase containing less than 40% acetonitrile. [Brief description of the drawings]

[0028]

Figure 1

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Figure 3

Figure 4

Figure 5

[0029] The aim of the method of the invention is to allow the identification of microorganisms in a short time, in particular less than 10 minutes, in an inexpensive process that can be used routinely without the need for highly qualified personnel.

[0030] In particular, the present invention relates to a method for identifying at least one microorganism present in a sample, based on the detection of peptides resulting from cleavage of a ribosomal protein of said microorganism, comprising the following steps: a) lysing microorganisms and cleaving proteins present in said sample to obtain a peptide mixture; b) decomplexing the peptide mixture using a liquid separation device connected to a mass spectrometer; c) atomizing the liquid eluted from the separation device using an ion source to generate an ion current; d) receiving, with the mass spectrometer, the ion current from the ion source and performing, for each cycle of a plurality of cycles, a series of filtering steps on the ion current to detect transitions, the transitions including a precursor ion and at least one fragment ion of the precursor ion, the transitions being read from a predefined transition list using the mass spectrometer, and for each transition in the series of transitions, the mass spectrometer selecting and fragmenting a precursor ion of each transition; e) receiving, with a processor, data regarding a plurality of transitions used to monitor the peptide mixture; f) using a processor, allocating the plurality of transitions into two or more consecutive transition groups and assigning them to the predefined transition list; g) monitoring, with a processor, at least one sentinel transition associated with a sentinel compound in each of two or more consecutive groups, wherein the at least one sentinel transition is selected as having the slowest predicted retention time in the group; h) when a signal of at least one sentinel transition of a group is detected by the mass spectrometer, using the processor to stop monitoring the transitions of the previous group while starting monitoring at least one sentinel transition of the next successive group; i) optionally using a processor to generate a chromatogram or electropherogram from the detection of transitions read from a predefined list by said mass spectrometer; wherein each transition read from the predefined list is associated with a peptide, and a microorganism is identified according to said peptide detected.

[0031] Each step of this identification method is described in more detail below.

[0032] In the sense of the present invention, the term "microorganism" means a bacterium or a yeast.

[0033] In a particular embodiment of the invention, the microorganism is a pathogenic microorganism that causes disease in humans. In particular, the microorganism is selected from the most common microorganisms that cause bacterial infections, sepsis, or urinary tract infections.

[0034] The microorganisms that can be identified by the method of the invention can be groups of microorganisms that represent a family, genus or species of pathogenic or non-pathogenic microorganisms.

[0035] For example, the following bacteria belonging to the ESKAPE group are examples of microorganisms that can be identified: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae (pneumonia bacillus) Klebsiella pneumoniae ), Acinetobacter baumannii ( Acinetobacter baumannii ), Pseudomonas aeruginosa, and · Enterobacter spp.

[0036] Other examples of identifiable microorganisms include: Yeasts, e.g. Candida krusei ( Candida krusei ), Streptococcus spp., e.g. Streptococcus pneumoniae ( Streptococcus pneumoniae ), Coagulase-negative staphylococci.

[0037] In the sense of the present invention, the term "sample" means a biological sample obtained from a mammal selected from the group consisting of blood, serum, lymph, mucus, odor, saliva, tracheobronchial aspirate, cerebrospinal fluid and urine; or A sample selected from the group consisting of used water, food, beverage, soil samples and surface samples. means...

[0038] The sample may contain one or more microorganisms. The method is adapted for the identification of at least one microorganism and can therefore be carried out for the identification of multiple microorganisms.

[0039] Advantageously, the method of the invention is achieved in a short time, in any case less than 10 minutes, and is therefore suitable for the diagnosis, in particular for the diagnosis of sepsis in humans.

[0040] In a preferred embodiment, the sample is a biological sample obtained from a human selected from the group consisting of blood, serum, lymph, mucus, malodor, saliva, tracheobronchial aspirate, cerebrospinal fluid and urine, in particular a blood sample.

[0041] Step (a) of this method Prior to any analytical step, microorganisms present in the sample are preferably pelleted by centrifugation, filtration, acoustophoresis, flotation or spinning.

[0042] In a particular embodiment of the invention, step (a) comprises a preliminary substep of removing peptides that do not result from cleavage of ribosomal proteins, which is achieved in particular by the addition of a detergent to the sample to be assayed.

[0043] To obtain a peptide mixture, lysis of the microorganisms present in the sample and cleavage of the proteins are carried out, in certain embodiments both operations being carried out in parallel.

[0044] Since the signatures of microorganisms are derived from proteins, it is necessary to treat the samples before analysis by mass spectrometry. To generate peptides from the proteins present in the sample, these proteins can be digested with protein decomplexing enzymes (proteases), such as trypsin or pepsin, or by the action of chemical reagents, such as cyanogen bromide (CNBr) or hydroxyl radical (H2O2) treatment.

[0045] However, cleavage of proteins by enzymatic digestion is preferred because it is easier to control, causes less denaturation of protein structure than treatment with chemical reagents, and is particularly specific.

[0046] Enzymatic digestion is the action of an enzyme (or enzymes) that allows the production of peptides from proteins under specific reaction conditions. Enzymes that cleave proteins at specific locations and thus effect protein decomplexation are called proteases. Each protease is usually able to recognize a specific cleavage site within the amino acid sequence.

[0047] International application WO2005 / 098071 describes proteases which may be cited as examples: Pepsin, which hydrolyzes peptide bonds at the level of the amine functions of aromatic amino acids (Tyr, Trp, Phe), which is used at acidic pH. Endolysine, which cleaves the peptide bond of the CO group of lysine; · Trypsin, which hydrolyzes peptide bonds at the level of the carboxyl groups of Lys and Arg residues.

[0048] In the method of the invention, cleavage of the protein is preferably carried out by digestion with the enzyme trypsin.

[0049] Advantageously, the incubation temperature during lysis and protein cleavage in step (a) is about 37°C.

[0050] The generation of the peptide mixture can be achieved by simple lysis or it can be sped up using various auxiliary processes such as pressure, microwave oven or even ultrasonic device. Lysis of cells present in the sample can be more efficient by using one of these three methods.

[0051] Step (b) Deconjugation of peptides refers to the partial step of separating the peptides, which is carried out by liquid separation techniques such as liquid chromatography or capillary electrophoresis.

[0052] Liquid chromatography (LC) is a separation technique in which the mobile phase is a liquid. It can be performed either in a column or on a plate. It includes high performance liquid chromatography (HPLC), normal phase liquid chromatography (NPLC) and reversed phase liquid chromatography (RPLC) among others.

[0053] Capillary electrophoresis (CE) is a set of separation techniques performed in sub-millimeter diameter capillaries and micro- and nanochannels, including capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), capillary isoelectric focusing (CIEF), capillary isotachophoresis and micellar electrokinetic chromatography (MEKC), among others.

[0054] In a preferred embodiment of the invention, the decomplexation step is carried out by reverse phase liquid chromatography.

[0055] In one embodiment of the invention, the step of decomplexing the peptide mixture is carried out on a reversed-phase column using a mobile phase containing less than 40% acetonitrile. The peptides are selected as a function of their sequence length, since this characteristic correlates with the retention factor k and therefore the percentage of acetonitrile required for elution from the reversed-phase column.

[0056] Acetonitrile (methyl cyanide) is a polar aprotic solvent.

[0057] Step (c) The method is carried out in a mass spectrometer connected to a liquid separation device. The mass spectrometric analysis is carried out in a targeted manner.

[0058] In general, mass spectrometry (MS) is an analytical method in which an ion source is used to ionize the liquid to be analyzed to generate an ion current. The "atomization" of a liquid containing a peptide mixture is well known to those skilled in the art.

[0059] In a preferred embodiment, the mass spectrometry is tandem mass spectrometry MS / MS, preferentially parallel reaction monitoring (PRM) or multiple reaction monitoring MRM.

[0060] In this embodiment, the transition is an MRM transition.

[0061] Step (d) The mass spectrometer receives the ion current from the ion source and, for each cycle of a plurality of cycles, performs a series of filtering steps on the ion current to detect transitions, the transitions including a precursor ion and at least one fragment ion of the precursor ion, the transitions being read from a predefined transition list using the mass spectrometer, and for each transition in the series of transitions, the mass spectrometer selects and fragments a precursor ion of each transition.

[0062] In the sense of the present invention, a "predefined transition list" means a finite list of transitions, i.e. specific pairs of m / z values ​​associated with precursor ions and fragment ions, each transition associated with a specific peptide. In other words, the mass spectrometer systematically monitors these transitions, each associated with a specific peptide and defined prior to the analysis.

[0063] This process does not analyze the precursor ion spectra in real time, nor does it add information to the list, such real time analysis is described for example in International Application WO2014 / 116711.

[0064] The predefined transition list was established based on Table 1, which regroups the 423 peptides shown below having the sequences SEQ ID NO:1 to SEQ ID NO:423.

[0065] This predefined list contains transitions that are each associated with a specific peptide that may be present in the peptide mixture being analyzed and are therefore monitored.

[0066] In a particular embodiment of the invention, the predefined list comprises at least one transition associated with a peptide exhibiting a peptide sequence selected from SEQ ID NO:1 to SEQ ID NO:423.

[0067] Advantageously, the predefined list comprises transitions associated with at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300 or 400 different peptides selected from the group consisting of peptides having a peptide sequence selected from SEQ ID NO:1 to SEQ ID NO:423.

[0068] In particular, the predefined list includes transitions associated with 423 peptides as listed in Table 1.

[0069] In another embodiment, the predefined list comprises transitions that are exclusively associated with at least one of the following groups of peptides: peptides specific for the order Enterobacteriaceae, in particular showing a sequence selected from SEQ ID NOs: 155-158, 168-180, 200-201, 223-229, 245-264, 274-277, 282-289, 306-307, 320-327, 393-395, 398-409 and 422-423; Acinetobacter specific peptides, in particular those having a sequence selected from SEQ ID NO: 13 to SEQ ID NO: 42: Enterococcus-specific peptides, in particular those having a sequence selected from SEQ ID NO: 181 to SEQ ID NO: 199; Candida-specific peptides, in particular those having a sequence selected from SEQ ID NO: 81 to SEQ ID NO: 150; peptides specific for sequences selected in particular from SEQ ID NO: 328 to SEQ ID NO: 345 and SEQ ID NO: 410 to SEQ ID NO: 421; peptides specific for the genus Streptococcus, in particular those having a sequence selected from SEQ ID NO: 349 to SEQ ID NO: 392; a peptide specific for Pseudomonas aeruginosa, in particular having a sequence selected from SEQ ID NO: 290 to SEQ ID NO: 300; or Other genus / species-specific peptides, in particular those having a sequence selected from SEQ ID NOs: 1 to 12, SEQ ID NOs: 43 to 80, SEQ ID NOs: 151 to 154, SEQ ID NOs: 159 to 167, SEQ ID NOs: 202 to 222, SEQ ID NOs: 230 to 244, SEQ ID NOs: 265 to 273, SEQ ID NOs: 278 to 281, SEQ ID NOs: 301 to 305, SEQ ID NOs: 308 to 319, SEQ ID NOs: 346 to 348 and SEQ ID NOs: 396 to 397.

[0070] Step (e) The generated plurality of transitions is data received by a processor and can be used to monitor the peptide mixture using said processor.

[0071] The term "processor" means, in the sense of the present invention, any digital circuit that performs operations on some external data source. In particular, the processor is a computer. In this case, the external data source is a mass spectrometer and the data to be transmitted are a number of transitions.

[0072] In the classical way, the computer is adapted to execute code instructions that perform part of the data processing. It may also include a data storage module (memory, e.g. flash), advantageously a user interface (typically a screen) and biometric acquisition means.

[0073] Steps (f), (g), (h) The plurality of transitions is assigned using a processor to two or more consecutive transition groups into the predefined transition list.

[0074] According to one embodiment, two or more consecutive transition groups are associated with a peptide group, each peptide being specific for a genus and / or species of a microorganism.

[0075] A step of monitoring at least one sentinel transition associated with a sentinel compound in each of two or more consecutive groups is then performed, and said at least one sentinel transition is selected, using the processor, as the one having the slowest predicted retention time in that group.

[0076] Sentinel compounds are discussed in more detail in the chapter devoted to them.

[0077] In a next step, when a signal of at least one sentinel transition of a group is detected by the mass spectrometer, a processor is used to stop monitoring the transitions of the previous group while starting monitoring at least one sentinel transition of the next successive group.

[0078] Optional Step (i) A chromatogram or electropherogram can be generated using a processor from the detection of transitions read from a predefined list by the mass spectrometer.

[0079] In this chromatogram or electropherogram, each peptide is represented by a peak that has been "reconstructed" from the data obtained in the mass spectrometer.

[0080] This step is not essential, but is useful for visual interpretation of the results.

[0081] Detected peptide Methods for the identification of microorganisms are based on the detection of peptides resulting from the cleavage of ribosomal proteins belonging to said microorganisms. Ribosomal proteins are known to be abundant and stable over time, i.e. not prone to mutations. These proteins are part of the ribosomes and in particular translate the genes encoded in the messenger RNA. There are two types of ribosomal proteins depending on the ribosomal subunit to which they belong: the letter L (large) stands for the large subunit and the letter S (small) for the small subunit.

[0082] In the method of the invention, each transition read from a predefined list is associated with a peptide, which is further represented by a peak on the compiled chromatogram or electropherogram.

[0083] As shown in the Examples section, microorganisms can be identified based on the peaks present in the chromatogram or electropherogram.

[0084] In one embodiment of the present invention, the detection of one peptide specific to the genus and / or species of the microorganism is sufficient to identify such a microorganism.

[0085] In another embodiment of the invention, the detection of at least two peptides specific for the genus and / or species of a microorganism is used to identify said microorganism.

[0086] Advantageously, the process of the invention allows the identification of two or more different species upon detection of two or more peptides, each one specific for a genus and / or species, present in the same sample.

[0087] Each transition is associated with a peptide, hereafter referred to as a "biomarker peptide", derived from a ribosomal protein of microbial origin that has been exhaustively selected according to the characteristics set out below.

[0088] First, these biomarker peptides must be specific to the genus and / or species of the microorganism being identified.

[0089] Second, the biomarker peptides must possess physicochemical properties that allow them to meet the specifications set for the liquid separation step, i.e. optimal peak capacity within short gradient times of a few minutes.

[0090] The peak capacity is defined as follows:

number

number

[0091] For example, in an experimental setup using a column length of 100 mm, an internal diameter of 1 mm, a particle size of 3.5 μm, a flow rate of 100 μL / min, and a shortened gradient time (4.12 min) to achieve a fast turnaround time, the peak capacity reaches an optimum as soon as the acetonitrile in the gradient solvent is 30-35%. Above this percentage, more peptides will be eluted at the same peak capacity, increasing the probability of interference in the signal associated with the target of interest.

[0092] Third, biomarker peptides were also selected as a function of sequence length, since this characteristic correlates with the retention factor k and therefore the percentage of acetonitrile required for elution from reversed-phase columns, especially from octadecyl reversed-phase columns. Thus, of all peptide candidates identified as species-specific biomarkers, only those containing 6–20 amino acids were ultimately retained in the identification assay, in order to keep acetonitrile in the gradient solvent below 40%.

[0093] In a preferred embodiment of the invention, the predefined list comprises at least one transition which relates to peptides comprising 6 to 20 amino acids and which is decomplexed during step (b) with a mobile phase comprising less than 40% acetonitrile.

[0094] More specifically, the predefined list includes at least one transition associated with a peptide exhibiting a peptide sequence selected from SEQ ID NO: 1 to SEQ ID NO: 423 as shown in Table 1 below.

[0095] Advantageously, each transition of the predefined list relates to a peptide selected from the group of peptides comprising or consisting of peptides having the sequences as depicted in SEQ ID NO:1 to SEQ ID NO:423.

[0096] In a particular implementation of this process, in the predefined list, at least one transition is associated with at least one peptide exhibiting a sequence selected from SEQ ID NO: 1 to SEQ ID NO: 423.

[0097] In another embodiment, the predefined list comprises transitions that are exclusively associated with at least one of the following peptide groups: peptides specific for the order Enterobacteriaceae, in particular showing a sequence selected from SEQ ID NOs: 155-158, 168-180, 200-201, 223-229, 245-264, 274-277, 282-289, 306-307, 320-327, 393-395, 398-409 and 422-423; Acinetobacter specific peptides, in particular those having a sequence selected from SEQ ID NO: 13 to SEQ ID NO: 42: Enterococcus-specific peptides, in particular those having a sequence selected from SEQ ID NO: 181 to SEQ ID NO: 199; Candida-specific peptides, in particular those having a sequence selected from SEQ ID NO: 81 to SEQ ID NO: 150; peptides specific for sequences selected in particular from SEQ ID NO: 328 to SEQ ID NO: 345 and SEQ ID NO: 410 to SEQ ID NO: 421; peptides specific for the genus Streptococcus, in particular those having a sequence selected from SEQ ID NO: 349 to SEQ ID NO: 392; a peptide specific for Pseudomonas aeruginosa, in particular having a sequence selected from SEQ ID NO: 290 to SEQ ID NO: 300; or Other genus / species-specific peptides, in particular those having a sequence selected from SEQ ID NOs: 1 to 12, SEQ ID NOs: 43 to 80, SEQ ID NOs: 151 to 154, SEQ ID NOs: 159 to 167, SEQ ID NOs: 202 to 222, SEQ ID NOs: 230 to 244, SEQ ID NOs: 265 to 273, SEQ ID NOs: 278 to 281, SEQ ID NOs: 301 to 305, SEQ ID NOs: 308 to 319, SEQ ID NOs: 346 to 348 and SEQ ID NOs: 396 to 397.

[0098] [Table 1] TIFF2024528623000004.tif215153TIFF2024528623000005.tif202156TIFF2024528623000006.tif216156 TIFF2024528623000007.tif211155TIFF2024528623000008.tif202156TIFF2024528623000009.tif205156 TIFF2024528623000010.tif208154TIFF2024528623000011.tif209154TIFF2024528623000012.tif222154 TIFF2024528623000013.tif207155TIFF2024528623000014.tif207153TIFF2024528623000015.tif204153 TIFF2024528623000016.tif211155TIFF2024528623000017.tif214155TIFF2024528623000018.tif204153 TIFF2024528623000019.tif209154TIFF2024528623000020.tif200153TIFF2024528623000021.tif205153 TIFF2024528623000022.tif201153TIFF2024528623000023.tif194155TIFF2024528623000024.tif176155 TIFF2024528623000025.tif191155TIFF2024528623000026.tif186155TIFF2024528623000027.tif178153

[0099] The method of the invention allows in particular the identification of microorganisms belonging to the following groups: Enterobacteriaceae Acinetobacter Enterococcus Candida Staphylococcus aureus, and Streptococcus spp.

[0100] The group of Enterobacteriaceae called Enterobacteriaceae includes 32 species: Citrobacter freundii, Citrobacter brachyura ( Citrobacter braakii ), Citrobacter koseri ( Citrobacter koseri ), Citrobacter yongae ( Citrobacter youngae ), Citrobacter worksmanii ( Citrobacter werkmanii ), Citrobacter portucalensis ( Citrobacter portucalensis ), Citrobacter clonae ( Citrobacter cronae ), Citrobacter amalonaticus ( Citrobacter amalonaticus ), Citrobacter farmeri ( Citrobacter farmer ), Citrobacter sedlachii ( Citrobacter sedlakii ), Citrobacter koseri ( Citrobacter koseri ), Enterobacter azubriae ( Enterobacter asburiae ), Enterobacter cloacae ( Enterobacter cloacae ), Enterobacter formakei ( Enterobacter hormachei ), Escherichia coli, Hafnia alvei ( Hafnia alvei ), Klebsiella aerogenes ( Klebsiella aerogenes ), Klebsiella oxytoca ( Klebsiella oxytoca ), Klebsiella pneumoniae, Morganella morganii ( Morganella _ morganii ), Pantoea agglomerans ( Pantoea agglomerans ), Proteus mirabilis ( Proteus mirabilis ), Proteus vulgaris ( Proteus vulgaris ), Proteus koranbae ( Proteus columbae ), Proteus pennellii ( Proteus penneri), Proteus terrae ( Proteus terrae ), Raoultella ornitholytica ( Raoultella ornithinolytica ), Salmonella enterica ( Salmonella enterica ), Serratia marcescens ( Serratia marcescens ), Providencia Rettgeri ( Providencia rettgeri ) and Providencia Stuarte ( Providencia stuartii ).

[0101] Peptides from this group of Enterobacteriaceae are common to all species in this group.

[0102] The Acinetobacter group, called Acinetobacter common, includes four Acinetobacter species: Acinetobacter baumannii, Acinetobacter ruofiii ( Acinetobacter Iwoffii ), Acinetobacter ursingii ( Acinetobacter ursingii ) and Acinetobacter pittii ( Acinetobacter pittii ).

[0103] This Acinetobacter common group peptide is common to all species in this group.

[0104] The Enterococcus group, called Enterococcus common, includes two Enterococci: Enterococcus faecium and Enterococcus faecalis ( Enterococcus faecalis ).

[0105] The peptides of this Enterococcus_common group are common to all species in this group.

[0106] The group of Candida species known as Candida common includes seven species: Candida albicans ( Candida albicans ), Candida auris (Candida auris ), Candida glabrata ( Candida glabrata ), Candida kefir ( Candida kefyr ), Candida krusei ( Candida krusei ), Candida tropicalis ( Candida tropicalis ) and Candida parasillosis ( Candida parapsilosis ).

[0107] The peptides of this Candida common group are common to all species in this group.

[0108] The Staphylococcus_coagulase_negative group includes 10 coagulase-negative staphylococci: Staphylococcus capitis ( Staphylococcus capitis ), Staphylococcus caprae ( Staphylococcus caprae ), Staphylococcus cohnii ( Stapylococcus cohnii ), Staphylococcus epidermidis ( Staphylococcus epidermidis ), Staphylococcus haemolyticus ( Staphylococcus haemolyticus ), Staphylococcus hominis ( Staphylococcus hominis ), Staphylococcus lugdunensis ( Staphylococcus lugdunensis ), Staphylococcus pettencolferi ( Staphylococcus pettenkorferi ), Staphylococcus saprophyticus ( Staphylococcus saprophyticus ), Staphylococcus warneri ( Staphylococcus warneri ).

[0109] This peptide of the Staphylococcus coagulase-negative group is common to all species in this group.

[0110] The Streptococcus_Common group includes 17 Streptococcus genera: Streptococcus agalactiae ( Streptococcus agalactiae ), Streptococcus dysgalactiae ( Streptococcus Dysgalactiae ), Streptococcus pneumoniae, Streptococcus pyogenes ( Streptococcus pyogenes ), Streptococcus gallolyticus subsp. gallolyticus ( Streptococcus Gallolyticus subsp. Gallolyticus ), Streptococcus pasteurianus (Streptococcus pasteurianus ) (Streptococcus gallolyticus subsp. pasteurianus), Streptococcus infantarius subsp. coli ( Streptococcus infantarius spp. Coli ) (or Streptococcus lutetiensis ( Streptococcus lutetiensis )), Streptococcus infantarius subsp. infantarius, Streptococcus mitis ( Streptococcus mitis ), Streptococcus oralis ( Streptococcus oralis ), Streptococcus salivarius ( Streptococcus salivarius ), Streptococcus anginosus ( Streptococcus anginosus ), Streptococcus parasanguinis ( Streptococcus parasanguinis ), Streptococcus constellatus ( Streptococcus constellatus ), Streptococcus sanguinis ( Streptococcus sanguine ), Streptococcus gordonii ( Streptococcus gordonii ), Streptococcus intermedius ( Streptococcus intermedius ).

[0111] The peptides of this Streptococcus_common group are common to all species in this group.

[0112] The Streptococcus bovis and related group includes four Streptococcus genera: Streptococcus gallolyticus subsp. gallolyticus, Streptococcus pasteurianus (Streptococcus gallolyticus subsp. pasteurianus), Streptococcus infantarius subsp. coli (or Streptococcus lutetiensis), and Streptococcus infantarius subsp. infantarius.

[0113] Peptides from the Streptococcus bovis and related groups are common to all species in this group.

[0114] The Streptococcus_Other_Streptococcus group includes nine genera of Streptococcus: Streptococcus mitis, Streptococcus oralis, Streptococcus salivarius, Streptococcus anginosus, Streptococcus parasanguinis, Streptococcus constellatus, Streptococcus sanguinis, Streptococcus gordonii, and Streptococcus intermedius.

[0115] This peptide of the Streptococcus_other_Streptococcus group is common to all species of this group.

[0116] mass spectrometer The method uses a mass spectrometer coupled to a liquid separation device and a processing means.

[0117] In certain embodiments of the invention, the mass spectrometer is a tandem mass spectrometer.

[0118] Tandem mass spectrometers are capable of multiple mass analyses, usually separated by some form of molecular fragmentation. Tandem MS can also be performed over time on a single mass spectrometer, such as a quadrupole ion trap. There are a variety of methods for fragmenting molecules for tandem MS, including collision-induced dissociation (CID), electron capture dissociation (ECD), electron transfer dissociation (ETD), infrared multiphoton dissociation (IRMPD), blackbody infrared radiation dissociation (BIRD), electron detachment dissociation (EDD), and surface-induced dissociation (SID).

[0119] In another embodiment of the invention, the mass spectrometer uses one of the following techniques: PRM (Parallel Reaction Monitoring), MRM (Multiple Reaction Monitoring), DIA (Data Independent Acquisition) or SWATH (Sequential Frame Acquisition of All Theoretical Fragment Ion Spectral Mass Analysis).

[0120] These techniques are well known to those skilled in the art.

[0121] The invention also relates to a mass spectrometer connected to a liquid separation device and adapted to carry out steps e), f), g) and h), in particular receiving data regarding a plurality of transitions used to monitor a peptide mixture; Allocating a plurality of transitions into two or more consecutive transition groups and assigning them to the predefined transition list; monitoring at least one sentinel transition in each of said two or more consecutive groups; - upon detection by the mass spectrometer of a signal of at least one sentinel transition of a group, starting monitoring of at least one transition of a next successive group; and In some cases, producing chromatograms or electropherograms The present invention also relates to a system for the implementation of the above method, comprising processing means adapted to said method.

[0122] In a classical way, the system includes a data processing module, i.e. a computer such as a processor, microprocessor, controller, microcontroller, FPGA, etc., which is adapted to execute code instructions for performing some of the data processing as indicated above, if necessary. This computer is adapted to execute code instructions for performing some of the data processing as indicated above.

[0123] The system also includes a data storage module (memory, eg flash), advantageously a user interface (typically a screen), and biometric acquisition means.

[0124] Sentinel Compound The present invention uses a technique described in patent EP3384517, referred to herein as the "Sentinel" acquisition mode, which allows for large multiplexing capabilities.

[0125] The methodology involves the detection of "sentinel compounds" by a tandem mass spectrometer, more precisely a method for triggering a group of multiple reaction monitoring (MRM) transitions from a series of successive groups when at least one sentinel transition of the group has been detected as part of the previous group, separating one or more compounds from the sample using a separation device; ionizing the one or more separated compounds received from the separation device using an ion source to generate an ion beam of one or more precursor ions; using a tandem mass spectrometer to receive an ion beam from an ion source, and for each cycle of a plurality of cycles, using the tandem mass spectrometer to perform on the ion beam a series of MRM precursor ion to product ion transitions read from the list, wherein for each transition in the series, the tandem mass spectrometer selects and fragments a precursor ion of each transition and mass analyzes a small mass-to-charge ratio (m / z) range around the m / z of the product ion of each transition to determine whether a product ion of each transition is detected; receiving, with a processor, a plurality of MRM transitions to be used for monitoring the sample. The method includes: dividing the plurality of MRM transitions into two or more consecutive MRM transition groups such that different groups can be separately monitored during the plurality of cycles using a processor; selecting, with the processor, at least one sentinel transition within each group of the two or more consecutive groups to be monitored that identifies a next group of the two or more consecutive groups; using a processor to input a first group of the two or more consecutive groups into a list of a tandem mass spectrometer; and if at least one sentinel transition of the first group is detected by the tandem mass spectrometer, using the processor to list a next group of two or more consecutive groups identified by the sentinel transitions. The present invention is characterized by further comprising:

[0126] The application of this method to microbial detection is described herein.

[0127] The sentinel compound for use in the method may be selected from the following compounds: the peptides resulting from the cleavage of the microbial protein in step (a), also called "endogenous peptides", in which case the method is hereinafter called the "sentinel-endogenous" method; peptides resulting from the self-cleavage (autolysis) of the trypsin enzyme or from the cleavage of proteins or peptides introduced into the sample, or exogenously introduced peptides, also called "exogenous peptides", in which case the method is hereinafter referred to as the "sentinel-exogenous" method; Other compounds.

[0128] Each implementation of the method is presented in more detail in the Examples section.

[0129] In the process of the present invention, in steps (g) and (h), a processor is used to monitor at least one sentinel transition associated with one sentinel compound; when a signal of at least one sentinel transition of a group is detected in the mass spectrometer, the processor is used to stop monitoring the transitions of the previous group while starting monitoring at least one sentinel transition of the next successive group.

[0130] Sentinel compounds are selected as those with the slowest expected retention times in their transition groups, in other words, the sentinel compounds are adapted, using the processor, to stop monitoring transitions associated with peptide groups with earlier retention times than the sentinel and start monitoring transitions associated with peptide groups with later retention times than the sentinel.

[0131] Peptide Group In another aspect, the present invention relates to a group of peptides adapted for carrying out the method as described above, said peptides originating from ribosomal proteins of microorganisms and containing between 6 and 20 amino acids, which are decomplexed on a reversed phase column, preferentially on an octadecyl reversed phase column, using a mobile phase containing less than 40% acetonitrile.

[0132] Furthermore, these peptides are specific to the genus and / or species of the microorganism and are therefore useful for its identification.

[0133] The group of peptides may include at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300 or 400 different peptides.

[0134] In a particular embodiment of the invention, the peptide group comprises at least one peptide exhibiting a peptide sequence selected from SEQ ID NO:1 to SEQ ID NO:423.

[0135] Advantageously, the peptide group comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300 or 400 different peptides selected from the group of peptides having peptide sequences selected from SEQ ID NO:1 to SEQ ID NO:423.

[0136] Particular peptide groups according to the invention are as follows: peptides specific for the order Enterobacteriaceae, in particular showing a sequence selected from SEQ ID NOs: 155-158, 168-180, 200-201, 223-229, 245-264, 274-277, 282-289, 306-307, 320-327, 393-395, 398-409 and 422-423; Acinetobacter specific peptides, in particular those having a sequence selected from SEQ ID NO: 13 to SEQ ID NO: 42: Enterococcus-specific peptides, in particular those having a sequence selected from SEQ ID NO: 181 to SEQ ID NO: 199; Candida-specific peptides, in particular those having a sequence selected from SEQ ID NO: 81 to SEQ ID NO: 150; Staphylococcus-specific peptides, in particular those exhibiting a sequence selected from SEQ ID NO: 328 to SEQ ID NO: 345 and SEQ ID NO: 410 to SEQ ID NO: 421; peptides specific for the genus Streptococcus, in particular those having a sequence selected from SEQ ID NO: 349 to SEQ ID NO: 392; a peptide specific for Pseudomonas aeruginosa, in particular having a sequence selected from SEQ ID NO: 290 to SEQ ID NO: 300; or Other genus / species-specific peptides, in particular those having a sequence selected from SEQ ID NOs: 1 to 12, SEQ ID NOs: 43 to 80, SEQ ID NOs: 151 to 154, SEQ ID NOs: 159 to 167, SEQ ID NOs: 202 to 222, SEQ ID NOs: 230 to 244, SEQ ID NOs: 265 to 273, SEQ ID NOs: 278 to 281, SEQ ID NOs: 301 to 305, SEQ ID NOs: 308 to 319, SEQ ID NOs: 346 to 348 and SEQ ID NOs: 396 to 397.

[0137] In certain embodiments, the group of peptides comprises all of the peptides listed in Table 1.

[0138] In another embodiment, the peptide group consists of 423 peptides as listed in Table 1.

[0139] These groups of peptides according to the invention can be used in any method or process for the identification of at least one microorganism in a sample.

[0140] The present invention also relates to the use of one of this group of peptides as defined above for the identification of at least one microorganism in a sample, in particular in a biological sample, more particularly in a human blood sample. EXAMPLES

[0141] Although the invention has been described herein with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of the principles and applications of the present invention. It is thus to be understood that numerous modifications can be made to the illustrative embodiments and other arrangements can be devised without departing from the spirit and scope of the invention as defined by the appended claims.

[0142] Example 1: Construction of a Sentinel-MRM method using endogenous peptides as group triggers To identify microorganisms, for example, endogenous peptides can be used as group triggers, i.e., sentinel compounds.

[0143] For example, a sentinel-MRM method can be constructed as follows.

[0144] The specific peptides of each microorganism can be divided into eight groups: a) Enterobacteriaceae group to identify 32 Enterobacteriaceae; b) Pseudomonas aeruginosa group for identifying Pseudomonas aeruginosa; c) the Staphylococcus aureus-Staphylococcus argenteus group to identify Staphylococcus aureus or Staphylococcus argenteus; d) Acinetobacter group for identifying four Acinetobacter species; e) Enterococcus group to identify two Enterococci; f) Candida group for identifying seven Candida species; g) the Streptococcus other group to identify 17 Streptococcus genera and 4 other species; h) an “other species” group for identifying 34 species including 10 coagulase-negative staphylococci.

[0145] Each group (except group h) contains peptides that are specific to each species in the group and peptides that are common to all species in the group.

[0146] For groups a, b, c, d, e and f, two sentinel peptides can serve as triggers for the group. These two sentinel peptides are strictly specific for the desired genus and common to all species of that group.

[0147] For example, in group a) Enterobacteriaceae, the sentinel peptides are two peptides that are common to all Enterobacteriaceae and are only present in Enterobacteriaceae species, meaning that they are not found in any other group.

[0148] Consider the example of a sample containing the pathogen Acinetobacter baumannii. In the Sentinel-MRM analysis of this sample, only group d) is triggered to monitor the transitions of Acinetobacter peptides. The group also contains peptides specific for Acinetobacter baumannii, resulting in a successful identification.

[0149] Figure 1 shows the chromatogram obtained as an example of the endogenous Sentinel-MRM method, where peptide intensities are expressed in arbitrary units.

[0150] The use of two peptides as sentinel compounds instead of just one is to ensure triggering of the transition group: if, for example, the peptide is mutated or absent in the analyzed sample, the second peptide can trigger the transition group.

[0151] Example 2: Construction of an exogenous sentinel-MRM method using an exogenous peptide (trypsin) as a group trigger In sample preparation, trypsin is used as a digestive enzyme. Therefore, trypsin is present in excess in all samples, and peptides resulting from autodigestion of trypsin itself can be used as sentinel compounds for the transition groups. The principle is to use the four peptides resulting from autodigestion of trypsin as triggers to distribute the specific peptides of each bacterial species (see Table 1) into four groups.

[0152] The FASTA sequence of trypsin (Accession No. P00761) (hereinafter referred to as SEQ ID NO: 428) is as follows: [ka]

[0153] The four peptides selected as sentinel triggers (sentinel compounds) are: NKPGVYTK (sequence number 424) VATVSLPR (SEQ ID NO: 425) LGEHNIDVLEGNEQFINAAK (SEQ ID NO: 426) IITHPNFNGNTLDNDIMLIK (sequence number 427)

[0154] FIG. 2 shows the distribution of these four peptides on the chromatogram.

[0155] One advantage of using trypsin-derived peptides is that all transitions of the selected peptides can be tracked, without the risk of a group not being triggered due to low intensity (e.g. low amounts of bacteria present in the sample) or due to interferences in the sample.

[0156] Another advantage of this implementation is that it allows for the identification of multiple bacteria in cases of, for example, multiple infections.

[0157] The performance of this method can also be easily augmented over the "endogenous" method with peptides from new species or species different from the panel selected for the present invention. For example, it is sufficient to create an MRM method with "new" peptides, know their retention times and place them in transition groups corresponding to the elution order of tryptic peptides.

[0158] Example 3: Identification of microorganisms from positive blood cultures by Sentinel-MRM mode acquisition 1. Isolation of Microorganisms from Positive Blood Cultures To isolate bacteria / yeasts present in blood culture flasks detected as positive, i.e. containing at least one microorganism, the procedure consists of lysing the hemocytes using a lysis buffer (here, 12% SDS) and then recovering the bacteria by centrifugation. Using a syringe and a 21G needle, take 1mL of blood culture medium and transfer it into a 1.5mL Eppendorf tube. Add 200 μL of 12% sodium dodecyl sulfate (SDS) and then vortex for 10 seconds. Centrifuge at 16100g for 2 minutes and remove the supernatant. Resuspend the pellet in 1 mL of physiological serum. Centrifuge at 16100g for 1 minute and discard the supernatant. Resuspend the pellet in 1 mL of physiological serum.

[0159] 2. Generation of Peptides by Enzymatic Digestion and Cell Lysis Add a scoop of glass beads (glass beads, acid washed, 150-212 μm, Sigma-Aldrich, ref G1145) to a 1.5 mL Eppendorf LowBind tube to a height of approximately 3-4 mm (one third of the final volume). After vortexing the sample prepared according to paragraph 1 for 10 minutes, take 200 µL and place it in the tube containing the glass beads. Prepare a 1 mg / mL trypsin solution in 150 mM ammonium bicarbonate buffer from lyophilized trypsin. Add 50 μL of freshly prepared trypsin solution (1 mg / mL) to the tube containing the beads and bacteria / yeast and vortex for 3 seconds. Place the sample in a sonicator, e.g., Diagenode water bath, set at 50°C. Immediately begin sonication for 1 min for 10 cycles. · Ultrasonic on for 30 seconds 30 seconds Ultrasonic off Ultrasonic output: Low After digestion, stop the reaction by adding 5 μL of formic acid and vortexing for 3 seconds. Centrifuge the tubes at 9600g (10,000rpm Accuspin Micro 17 bench tip centrifuge) for 5 minutes. Transfer 150 μL of the supernatant to an amber vial with an insert.

[0160] 3. Analytical conditions: Chromatography and mass spectrometry conditions After each sample has been treated according to the protocols in paragraphs 1 and 2, a 5 μL volume of digested protein is injected and analyzed under the following conditions: · HPLC equipment Agilent Pump 1290 (AGILENT, Santa Clara, USA) WATERS chromatography column (WATERS, Saint-Quentin-en-Yvelines, France) XBridge Peptide BEH C18, inner diameter 1 mm, length 100 mm, particle size 3.5 μm, pore size 130 Å) Solvent A: H2O 99.9% + 0.1% formic acid Solvent B: 99.9% acetonitrile + 0.1% formic acid Column oven temperature: 60℃ The HPLC gradient is defined according to Table 2 shown below.

[0161] [Table 2]

[0162] The peptide deconjugation step (ie, partial separation) is carried out under conditions using less than 40% solvent B, which is composed primarily of the polar solvent acetonitrile.

[0163] The eluate coming from the chromatography column is directly infused into the ionization source of a QTRAP® 6500+ mass spectrometer from AB SCIEX (Framingham, Mass., USA).

[0164] Other instrument settings are summarized in Table 3 below.

[0165] [Table 3]

[0166] Peptides obtained from tryptic digestion of bacterial proteins are analyzed by mass spectrometry in Sentinel-MRM mode. The peptides tracked and detected are specific for the microorganisms and therefore allow their identification.

[0167] 4. Identification of microorganisms by Sentinel-MRM acquisition mode, using endogenous peptides such as Sentinel, and application to blood culture samples (blind identification) The Sentinel-MRM method described in Example 1 is applied to blood culture samples.

[0168] Sentinel-MRM acquisition methods use 3 or more transitions for each peptide (see acquisition methods in Table 4), which consists of methods containing over 1500 transitions. To increase the specificity of the trigger sentinels and thus avoid group triggering due to interferences present in the sample, it was determined that alignment of 3 transitions is necessary for group triggering.

[0169] The obtained chromatogram is shown in Figure 3. The Enterococcus group was successfully triggered, and only peptides specific for the genus Enterococcus and the species Enterococcus faecium were detected. This result was confirmed by MALDI-TOF.

[0170] 5. Identification of Microorganisms by Sentinel-MRM Acquisition Mode, Using Trypsin-Generated Peptides as Sentinel Compounds The same samples as in the above examples were analyzed by applying the Sentinel-MRM acquisition method with tryptic peptides as the Sentinel trigger transition group.

[0171] The results are shown in Figure 4. Only peptides specific to the genus Enterococcus and Enterococcus faecium species were detected. This result was confirmed by MALDI-TOF.

[0172] Example 4: Analytical validation of the two acquisition methods in comparison with analysis by MALDI-TOF To validate the two acquisition methods, 42 samples were analyzed and the results are shown in Table 4.

[0173] [Table 4] TIFF2024528623000032.tif188158TIFF2024528623000033.tif203157TIFF2024528623000034.tif95157

[0174] Other tests are also being performed on 264 positive blood culture (bacteria or yeast) samples taken from patients.

[0175] The results obtained with the process of the present invention were compared with those obtained with the MALDI-TOF MS identification technique, and 100% correlation of the results was observed.

[0176] In terms of identification rate, the process of the present invention allowed the identification of at least one bacteria or yeast species in 93% of the samples assayed. No identification was obtained in 7% of the samples, likely due to insufficient amounts of microorganisms in the samples or the samples not actually containing any microorganisms.

[0177] Example 5. Identification of two bacterial species present in a sample by trypsin method Positive blood samples were analyzed by applying the Sentinel-MRM acquisition method with tryptic peptides as the sentinel trigger transition group.

[0178] The results are shown in Figure 5. Peptides specific to the Streptococcus bovis group (SEQ ID NOs: 365 and 367) and Escherichia coli species (SEQ ID NOs: 200 and 201) were detected.

[0179] This result was confirmed by MALDI-TOF the day after an overnight subculture step of the blood samples.

[0180] Advantageously, the process of the present invention allows the identification of at least two different species in the same sample.

[0181] References: In the order of citation in the text Patent Literature ·WO 2011 / 045544 · WO 2012 / 143535 ·WO 2012 / 143534 EP 3384517 WO 2005 / 098071 ·WO 2014 / 116711 References ·Holland, RD, Duffy, CR, Rafii, F., Sutherland, JB, Heinze, TM, Holder, CL, Voorhees, KJ, Lay, JO, Jr., 1999. Identification of bacterial proteins observed in MALDI TOF mass spectra from whole cells. Analytical chemistry. 71, 3226-3230 ·Lasch P, Schneider A, Blumenscheit C, Doellinger J. Identification of Microorganisms by Liquid Chromatography-Mass Spectrometry (LC-MS1) and in Silico Peptide Mass Libraries. Mol Cell Proteomics. 2020 Dec;19(12):2125-2139. doi: 10.1074 / mcp.TIR120.002061. Epub 2020 Sep 30. PMID: 32998977; PMCID: PMC7710138. ·Boulund F, Karlsson R, Gonzales-Siles L, Johnning A, Karami N, Al-Bayati O, Ahren C, Moore ERB, Kristiansson E. Typing and Characterization of Bacteria Using Bottom-up Tandem Mass Spectrometry Proteomics. Mol Cell Proteomics. 2017 Jun;16(6):1052-1063. ·Christian Blumenscheit, Yvonne Pfeifer, Guido Werner, Charlyn John, Andy Schneider, Peter Lasch, Joerg Doellinger. Unbiased antimicrobial resistance detection from clinical bacterial isolates using proteomics. bioRxiv 2020.11.17.386540; doi:

Claims

1. A method for identifying at least one microorganism present in a sample based on the detection of peptides resulting from the cleavage of ribosomal proteins of said microorganism, the following steps: a) lysing the microorganism and cleaving the proteins present in said sample to obtain a peptide mixture; b) using a liquid separation device connected to a mass spectrometer to decomplexify said peptide mixture; c) atomizing the liquid eluted from said separation device using an ion source to generate an ion current; d) using said mass spectrometer to receive said ion current from said ion source and, for each cycle of a plurality of cycles, performing a series of filtering steps for detecting transitions on said ion current, said transitions comprising a precursor ion and at least one fragment ion of said precursor ion, said transitions being read from a predefined transition list using a mass spectrometer, and for each transition of said series of transitions, the mass spectrometer selecting and fragmenting the precursor ion of each transition; e) using a processor to receive data regarding a plurality of transitions used to monitor the peptide mixture; f) using a processor to assign said plurality of transitions to two or more consecutive transition groups and assign them to said predefined transition list; g) using a processor to monitor at least one sentinel transition associated with one sentinel compound in each of two or more consecutive groups, said at least one sentinel transition being selected as having the slowest predicted retention time in that group; h) when a signal of at least one sentinel transition of a group is detected by the mass spectrometer, using a processor to stop monitoring the transitions of the previous group while starting to monitor at least one sentinel transition of the next consecutive group; i) In some cases, the method includes creating a chromatogram or an electropherogram from the detection of transitions read from a list predefined in the mass spectrometer using a processor, where each transition read from the predefined list is associated with a peptide, and a microorganism is identified according to the detection of the peptide.

2. The method according to claim 1, wherein the step of decomplexing the peptide mixture is performed by liquid chromatography or capillary electrophoresis.

3. The method according to claim 1 or 2, wherein the step of decomplexing the peptide mixture is performed using a mobile phase containing less than 40% acetonitrile.

4. The method according to claim 1 or 2, wherein the mass spectrometer is a tandem mass spectrometer.

5. The method according to claim 1 or 2, wherein the mass spectrometer uses PRM (parallel reaction monitoring), MRM (multiple reaction monitoring), DIA (data-independent acquisition), or SWATH MS (sequential window acquisition of all theoretical fragment ion spectra mass spectrometry).

6. The method according to claim 1 or 2, including a preliminary step of removing peptides not resulting from the cleavage of ribosomal proteins by adding a surfactant to the sample.

7. The method according to claim 1 or 2, wherein two or more consecutive transition groups are associated with a peptide group, and each of the peptides is specific to a genus and / or species of microorganism.

8. The method according to claim 1 or 2, wherein the cleavage of the protein is performed by digestion with trypsin enzyme.

9. The method according to claim 1 or 2, wherein the predefined list is associated with peptides containing 6 to 20 amino acids and includes at least one transition decomplexed with less than 40% acetonitrile in step b).

10. The method according to claim 1 or 2, wherein the predefined list includes at least one transition associated with a peptide showing a sequence selected from SEQ ID NO: 1 to SEQ ID NO:

423.

11. The method according to claim 1 or 2, wherein the sentinel compound is selected from the following group of compounds: peptides resulting from the cleavage of microbial proteins in step (a), peptides resulting from the autoproteolysis of trypsin enzyme, peptides resulting from the cleavage of proteins or peptides introduced into the sample, exogenously introduced peptides, and other compounds.

12. The sample is ・A biological sample obtained from a mammal, selected from the group consisting of blood, serum, lymph, mucus, body odor, saliva, tracheobronchial aspirate, cerebrospinal fluid and urine, or ・A sample selected from the group consisting of used water, food, beverage, soil sample and surface sample, the method according to claim 1 or 2.

13. A mass spectrometer connected to a liquid separation device, and adapted for carrying out steps e), f), g) and h), in particular ・Receiving data regarding a plurality of transitions used for monitoring a peptide mixture, ・Assigning the plurality of transitions to two or more consecutive transition groups and assigning them to the predefined transition list, ・Monitoring at least one sentinel transition in each of the two or more consecutive groups, ・Starting the monitoring of at least one transition of the next consecutive group when a signal of at least one sentinel transition of a group is detected by the mass spectrometer, and ・Optionally, a system for implementing the method according to claim 1 or 2, comprising processing means adapted to create a chromatogram or an electroferrogram.

14. A group of peptides suitable for implementing the method according to claim 1 or 2, wherein the peptides result from ribosomal proteins, contain 6 to 20 amino acids and are decomplexed using a mobile phase containing less than 40% acetonitrile.

15. A group of peptides according to claim 14, comprising at least one peptide showing a peptide sequence selected from SEQ ID NO: 1 to SEQ ID NO: 423.

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