Method for identifying esbl enzymes in enterobacteria

Abstract

The present invention relates to a method for determining the properties of resistance to beta-lactam antibiotics of a strain of enterobacteria present in a biological sample, which method comprises the steps of: a) lysis of the bacteria and hydrolysis of the bacterial proteins in order to obtain a mixture of peptides, b) analysis of this mixture of peptides by targeted mass spectrometry coupled with reverse phase liquid chromatography, detection of at least one specific peptide of an enzyme exhibiting extended spectrum beta-lactamase (ESBL) activity being indicative of the resistance to beta-lactams of the strain of enterobacteria present in the biological sample, the method being characterised in that the analysis step (b) is carried out in the presence of an ion pairing reagent (IPR) added into the chromatography column or into the mixture of peptides.

Description

[0001] Method for identifying ESBL enzymes in enterobacteria

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to an analytical method using reverse-phase liquid chromatography coupled with mass spectrometry (LC-MS / MS) to detect extended-spectrum β-lactamase (ESBL) activities linked to the presence of certain enzymes produced by enterobacteria.

[0004] STATE OF THE ART

[0005] Sepsis refers to any generalized inflammatory reaction associated with a serious bacterial infection. According to the Pasteur Institute, a person dies of sepsis every 5 seconds worldwide. In France, this infection is fatal for 27% of patients, and this figure can rise to more than 50% in cases of septic shock. In 2017, the WHO classified sepsis as a public health priority and published a list of pathogens that should be studied as a priority for determining new therapeutic approaches; this list notably includes the bacteria Acinetobacter baumannii, Pseudomonas aeruginosa, and enterobacteria producing carbapenemase or enzymes with extended-spectrum β-lactamase (ESBL) activity.

[0006] Currently, different families of antibiotics are used to treat sepsis in hospitals. One of the best known and most widely used is the B-lactam family, which includes penicillins, cephalosporins, monobactams, and carbapenems. B-lactams work by inhibiting the synthesis of the bacterial cell wall by blocking the production of peptidoglycan. These molecules share a common structure, corresponding to the presence of a four-atom ring, the B-lactam, which is their active site.

[0007] Extended-spectrum β-lactamase (ESBL) enzymes

[0008] There are so-called "resistance" mechanisms that prevent the action of these antibiotics: in particular efflux systems, the impermeability of the bacterial cell wall, and B-lactamases. These enzymes inactivate B-lactams by hydrolyzing their B-lactam ring. The first TEM B-lactamase, which hydrolyzes ampicillin and first-generation cephalosporins, was identified in the bacterium Escherichia coli. To address this problem, new extended-spectrum antibiotics have been developed, but their overuse in humans and animal husbandry has led to the emergence of extended-spectrum B-lactamases (ESBLs).

[0009] The Ambler and Bush-Jacoby-Medeiros classifications are the two existing classifications for β-lactamases. The Ambler scheme proposes a molecular classification and divides these enzymes into 4 classes: A, B, C and D. Those in class A, C and D are said to be serine active while those in class B are called metallo-β-lactamases (carbapenemases). With the exception of OXA-type ESBLs (class D), ESBLs are class A β-lactamases.

[0010] The Bush-Jacoby-Medeiros classification allows these enzymes to be classified according to their functional groups. This classification was updated in 2010 and divides β-lactamases into 3 groups: 1, 2 and 3 (Bush & Jacoby, 2010).

[0011] • Group 1 enzymes are cephalosporinases that belong to molecular class C. They are carried by plasmids in many Enterobacteriaceae and some other organisms, and are generally resistant to inhibition by clavulanic acid and active against cephamycins (e.g., cefoxitin). They have a high affinity for aztreonam, unlike class A cephalosporinases. Enzymes belonging to this subgroup are CMY, MIR, MOX, LAT, FOX, DHA, ACT, ACC, and CFE.

[0012] • Group 2 enzymes represent the largest group of β-lactamases. Enzymes in subgroups 2a, 2b, 2be, 2br, 2ber, 2c, 2ce, 2e, and 2f belong to molecular class A, and those in subgroups 2d, 2de, and 2df belong to molecular class D. o Subgroup 2a includes penicillinases that are predominantly chromosomal. They are the predominant β-lactamases of Gram-positive bacteria such as staphylococci and occasionally enterococci. These enzymes hydrolyze benzylpenicillin and many penicillin derivatives, but with difficulty hydrolyze cephalosporins, carbapenems, and monobactams. These enzymes are inhibited by clavulanic acid and tazobactam. This subgroup notably contains the enzyme PC-1. o Subgroup 2b includes penicillinases and cephalosporinases.These enzymes readily hydrolyze first-generation penicillins and cephalosporins and are strongly inhibited by clavulanic acid and tazobactam. TEM-1, TEM-2, and SHV-1 are included in this subgroup. o Subgroup 2br includes enzymes that hydrolyze first-generation penicillins and cephalosporins and are resistant to clavulanic acid and tazobactam. To date, 2br includes variants of TEM (e.g., TEM-30) and SHV (e.g., SHV-10). o Subgroup 2be includes ESBLs. These enzymes hydrolyze penicillins, first- to fourth-generation cephalosporins, monobactams, and additionally hydrolyze one or more oxyimino-B-lactams, such as cefotaxime, ceftazidime, and aztreonam. In general, ESBL-producing strains remain susceptible to cephamycins, carbapenems, and inhibition by clavulanic acid.This compound is therefore sometimes administered in combination with β-lactam antibiotics to limit the action of resistance enzymes. This subgroup includes the enzymes TEM-3, SHV-2, and CTX-M-15. o Subgroup 2ber includes TEM enzymes with ESBL-like activity and resistance to clavulanic acid and tazobactam. These enzymes are also called β-lactamase CMT (TEM mutant complex). TEM-50 is part of subgroup 2ber. o Subgroup 2c contains penicillinases that can hydrolyze carbenicillin. Clavulanic acid or tazobactam can inhibit these enzymes. PSE-1 and CARB-3 are part of subgroup 2c. o Subgroup 2ce contains carbenicillinase with broad activity against cefepime and cefpirome. RTG-4 is part of this subgroup. o Subgroup 2d includes β-lactamases that hydrolyze oxacillin. These enzymes are known as OXA enzymes and are variably inhibited by clavulanic acid.Enzymes found in this subgroup include OXA-1 and OXA-10. o Subgroup 2de includes OXA enzymes with an extended spectrum that includes oxyimino-B-lactams but not carbapenems. OXA-11 and OXA-15 are part of subgroup 2de. o Subgroup 2df contains OXA enzymes with carbapenem hydrolysis activities. OXA-23 and OXA-48 are part of subgroup 2df. o Subgroup 2e contains extended-spectrum cephalosporinases and are inhibited by clavulanic acid or tazobactam. One enzyme found in this group is CepA. o Subgroup 2f contains carbapenemases of molecular class A. These enzymes are better inhibited by tazobactam than by clavulanic acid. KPC-2, IMI-1 and SME-1 are enzymes that belong to subgroup 2f.

[0013] • Group 3 enzymes are carbapenemases that belong to molecular class B. They are resistant to inhibition by clavulanic acid and tazobactam. Enzymes that belong to this subgroup are IMP-1, VIM-1, CcrA,

[0014] I ND-1, L1, CAU-1, GOB-1, FEZ-1, CphA and Sfh-1.

[0015] ESBLs of the TEM and SHV type belong to the 2be and 2ber subgroups, i.e. they are capable of hydrolyzing penicillins, cephalosporins of 1 ère at 4 ème generation and monobactams.

[0016] In general, ESBL-producing strains remain susceptible to cephamycins and carbapenems. Furthermore, ESBLs of the 2be subgroup are inhibited by clavulanic acid. This compound is therefore sometimes administered in combination with B-lactam antibiotics to limit the action of resistance enzymes.

[0017] Clinical consequences of the existence of these ESBL enzymes inducing resistance to 8-lactam antibiotics

[0018] Enzymes with ESBL activity, hereinafter referred to as "ESBL", are increasingly found in blood infections in patients.

[0019] These enzymes are induced by plasmids of gram-negative bacteria, mostly resulting from mutations of natural beta-lactamases such as TEM-1, TEM-2 or SHV-1.

[0020] ESBLs are a major public health concern. They induce a high level of resistance to antibiotics, including third-generation cephalosporins. Sepsis is already characterized by a high mortality rate that increases rapidly over the hours without appropriate treatment. After 5 hours without appropriate treatment, the patient survival rate has already halved. It is only 10% after 24 hours and drops to less than 5% after 36 hours.

[0021] When sepsis is suspected in a patient, a blood sample is taken and cultured (blood culture). If bacteria are present in the collected sample, they will multiply over the hours until they reach a sufficient concentration for the sample to be declared positive. It takes at least 6 to 8 hours for a blood culture to be declared positive and another 6 hours to identify the bacteria. The assessment of potential antibiotic resistance is the longest step to perform, and sometimes requires up to 24-48 hours to obtain the results. However, it is common to start antibiotic treatment before obtaining these results, because such a long period without treatment would lead to a very low patient survival rate.

[0022] The solution used in hospitals to overcome the lengthy methods of identifying the bacteria and assessing resistance mechanisms is to give the patient extended-spectrum antibiotics and then adjust the treatment over time. However, this choice involves various subsequent problems, including an increase in antibiotic resistance in the population. Current techniques for detecting and identifying ESBLs

[0023] The development of rapid, efficient and robust analytical methods is essential in diagnosis in order to be able to offer the appropriate treatment to each patient as soon as possible.

[0024] Currently, routine ESBL detection methods are limited and require several dozen hours to obtain an antibiogram. They are mainly based on the use of diffusion disks, in particular the synergy test between the mixture of amoxicillin and clavulanic acid (AMC), and a C3G (for example ceftazidime) or aztreonam. Automated bacteriology analyzers (Vitek 2®, Alfred 60AST®, Mini-Api®, Phoenix®, Microscan®) are increasingly used because they are faster; for example, VITEK® 2 can provide identification and antibiogram results in less than 5 hours.

[0025] However, the sensitivity and specificity of these machines are not always sufficient and may require additional tests.

[0026] Numerous molecular biology studies using PCR (Polymerase Chain Reaction) amplification followed by sequencing have been tested. Real-time PCR and direct pyrosequencing of the products can notably allow the identification of ESBL-like CTX-M variants. Pyrosequencing is performed using cycles of denaturation, annihilation and extension of the chain, followed by sequencing. However, the use of PCR has disadvantages such as the high cost of the probes, as well as the lack of information on the actual expression of the proteins involved in resistance mechanisms.

[0027] In response to the current need for detection and evaluation of ESBL expression levels, new analytical strategies are beginning to emerge to rapidly determine the resistance profiles of bacteria.

[0028] Following the first studies concerning the identification of bacteria by MALDI-TOF (Matrix Assisted Laser Desorption Ionization - Time Of Flight), mass spectrometry is increasingly used.

[0029] MALDI-TOF has been used as a routine bacteriological analysis method in hospitals since its implementation ten years ago. This technique has reduced the time required for bacterial identification, particularly in the case of bloodstream infections. MALDI-TOF requires less data reprocessing time than conventional biochemical analysis methods in terms of identification. Therefore, the use of this analytical technique has rapidly spread in hospital laboratories and has become a technique of choice for routine testing in clinical microbiology.

[0030] In recent years, various studies have highlighted the possibility of using MALDI-TOF to identify the mechanism of antibiotic resistance and the activity of β-lactamases (Oviano et al., 2014). Some studies also use MALDI-TOF to perform mini-sequencing, particularly in the field of antibiotic resistance to extended-spectrum β-lactamases (ESBL).

[0031] Despite its effectiveness, MALDI-TOF is still not used as a routine method for the characterization of ESBLs. Indeed, for the Enterobacteriaceae group in particular, differentiation between certain resistance mechanisms is not always possible, as they present a remarkably similar profile (Hou et al., 2019).

[0032] Two types of approaches are generally used to study proteins by mass spectrometry: top-down proteomics and bottom-up proteomics.

[0033] Top-down proteomics allows for the direct analysis of proteins through the use of high-resolution mass spectrometry (HRMS) such as Orbitrap technology, powerful software, and extensive databases. This technology is particularly suitable for high-throughput screening analyses and can be used in targeted approaches as well as for quantitative analyses. However, the difficulty of obtaining an accurate understanding of this instrumentation and its high cost explain the limitations of its application in a routine setting.

[0034] In contrast, bottom-up mass spectrometry is a more affordable technique based on peptide analysis by mass spectrometry, most often at low resolution (LRMS). LC-MS / MS is particularly promising and allows the separation and identification of peptides in bacteriology. Peptides are obtained by enzymatic digestion of proteins. This approach is easier to apply in hospital laboratories because it is simpler to use, more affordable, and less time-consuming in terms of reprocessing.

[0035] Unlike top-down proteomics, bottom-up proteomics does not cover the entire protein sequence. It is therefore necessary to choose the best candidate peptides from the complete sequence, i.e. those particularly specific to an ESBL, which will be characterized by mass spectrometry to precisely detect and identify the ESBL(s) present in the sample studied.

[0036] The article by (Fleurbaaij et al., 2017) presents a method for identifying beta-lactamase-resistant bacteria using MS / MS mass spectrometry coupled with liquid chromatography. The samples processed are cultured blood samples identified as "positive", i.e., containing a bacterial population of the species E. coli and / or K. pneumoniae.

[0037] US patent application 2017 / 0052198 describes a method for detecting cephalosporin (beta-lactamase enzyme) resistance based on the detection of specific peptides by MS / MS mass spectrometry coupled with liquid chromatography. The peptides are first obtained by enzymatic digestion.

[0038] Patent EP 2 699 692 details a low-resolution RPLC-MS / MS analysis strategy for detecting at least one cephalosporin resistance mechanism. Different resistance mechanisms are sought, such as resistance to TEM-type B-lactams in strains of Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Enterobacter aerogenes, and other species capable of producing B-lactamases. This method indicates that "only the portion of the gradient between 3 and 34 minutes is useful for analysis."

[0039] However, several ESBL-specific peptides cited in this patent elute before 3 minutes. For example, the SGASER peptide (SEQ ID NO. 7) found in some TEM variants elutes at 1.4 minutes, and therefore cannot be identified with the method as described.

[0040] It was noted by the inventors that certain short sequence peptides, consisting of 5 or 6 amino acids, are very poorly retained in reverse phase, which is a real obstacle to their detection by LC-MS / MS.

[0041] The present invention provides a solution to this problem of non-detection of certain peptides specific to ESBLs, due to their short sequence and therefore their non-retention on reverse-phase chromatography columns, by ascending mass spectrometry.

[0042] BRIEF STATEMENT OF THE INVENTION

[0043] The present invention relates to a method for determining the properties of resistance to beta-lactam antibiotics of an enterobacterium strain present in a biological sample, comprising the following steps: a) lysis of the bacteria and hydrolysis of the bacterial proteins, to obtain a mixture of peptides, b) analysis of this mixture of peptides by targeted mass spectrometry coupled with reverse-phase liquid chromatography, the detection of at least one peptide specific for an enzyme with extended-spectrum beta-lactamase (ESBL) activity being indicative of the resistance to beta-lactams of the enterobacterium strain present in said biological sample, said method being characterized in that the analysis step (b) is carried out in the presence of an ion-pairing agent (IPR) added to the chromatography column or to the mixture of peptides.

[0044] The present invention also relates to a kit for its use in carrying out the method described above, said kit comprising trypsin and heptafluorobutyric acid. DESCRIPTION OF THE FIGURES

[0045] Figure 1. Chromatogram illustrating the detection of the peptide SGÀGK (SEQ ID NO. 8) in sample No. 27. The retention time of this peptide is 2.2 minutes, under the conditions of the method of the invention.

[0046] Figure 2. Impact of the concentration of the HFBA solution injected upstream of the analysis to increase the retention time of said peptides on the stationary phase, i.e. to slow down their elution with the mobile phase. The percentage of HFBA tested is between 0 and 0.5%. The retention time for each peptide tested is indicated in minutes. 7 peptides with a short sequence (less than 7 residues) and therefore presenting very little retention in reversed phase were used: SGASER (SEQ ID NO.7), SGAGER (SEQ ID NO.21), TGASER (SEQ ID NO.25), TGAGER (SEQ ID NO.33), SGAGK (SEQ ID NO.8), TGASK (SEQ ID NO.26), and SGTGK (SEQ ID NO.9).

[0047] Figure 3. Impact of HFBA addition on the area under the chromatographic peaks of the different monitored peptides containing 5 or 6 amino acids. HFBA is injected just upstream of the LC-MS / MS analysis using an injection program. The areas were normalized by a test analysis without HFBA where the peptides co-elute at the dead time. The drastic drop in the area under the peak curve (between 30 mM and 38 mM) indicates significant ion suppression during the co-elution of the peptides of interest with HFBA.

[0048] Figure 4. Chromatogram showing the HFBA signal at 30 mM injection. The x-axis is graduated in time units (minutes). The y-axis corresponds to the signal intensity detected by MS / MS.

[0049] DETAILED DESCRIPTION OF THE INVENTION

[0050] The invention presented here allows the characterization, in less than one hour, from a positive blood culture, of the resistance profile of enterobacteriaceae strains which express enzymes with ESBL activity of the TEM or SHV type.

[0051] This rapid detection of the resistance profile of the enterobacteria present in the sample allows the appropriate treatment to be administered quickly to each patient suffering from a bacterial infection, particularly sepsis.

[0052] The present invention relates more particularly to an improvement in the technology for detecting enzymes with ESBL activity in a sample, as described in patent EP 2 699 692.

[0053] Indeed, the analysis technique described in patent EP 2 699 692 does not allow the characterization of 20% of TEM-type ESBL enzymes and 90% of SHV-type ESBL enzymes, because peptides specific to these ESBL enzymes cannot be detected in the samples by the method considered (calculations carried out using the bldb.eu website).

[0054] A solution to this problem is proposed here. It consists of increasing the retention time of peptides not visualized in the method described in EP 2 699 692, due to their short sequence, by using an ion pairing agent (IPR) during the analysis.

[0055] Thus, the present invention relates to a method for determining the properties of resistance to beta-lactam antibiotics of a strain of enterobacterium present in a biological sample, comprising the following steps: a) lysis of the bacteria and hydrolysis of the bacterial proteins, to obtain a mixture of peptides, b) analysis of this mixture of peptides by targeted mass spectrometry coupled with reverse-phase liquid chromatography, the detection of at least one peptide specific for an enzyme with extended-spectrum beta-lactamase (ESBL) activity being indicative of the resistance to beta-lactams of the strain of enterobacterium present in said biological sample, said method being characterized in that the analysis step (b) is carried out in the presence of an ion-pairing agent (IPR) added to the chromatography column or to the mixture of peptides.

[0056] The terms used in this application are presented below to better define the invention.

[0057] Beta-lactam antibiotics are antibiotics whose molecular structure contains a beta-lactam nucleus. This large family of compounds includes penicillin derivatives, cephalosporins, monobactams, carbapenems, and β-lactamase inhibitors.

[0058] For the purposes of the invention, an enterobacterium strain designates a strain of the Enterobacteriaceae family. This large family of gram-negative bacteria includes in particular the following pathogenic species: Salmonella, Escherichia coli, Klebsiella, Shigella, Enterobacter and Citrobacter.

[0059] For the purposes of the invention, an “enzyme with extended-spectrum beta-lactamase (ESBL) activity” means a bacterial enzyme capable of inactivating at least one β-lactam by hydrolyzing its β-lactam ring. These enzymes with ESBL activity are also referred to in the present application as “variants”, because they are derived from enzymes that do not have this ESBL activity. Two types are particularly known: the TEM and SHV enzymes.

[0060] For the purposes of the invention, an "ion-pairing reagent", also referred to by its acronym IPR (ion-pairing reagent), denotes a compound for reducing ionic interactions between peptides and the hydrophobic stationary phase of chromatography. Many IPRs are known, such as formic acid, perfluorinated acids such as trifluoroacetic acid (TFA), pentafluoropropionic acid (PFPA) and heptafluorobutyric acid (HFBA), as well as the compounds n-butyl, n-pentyl, n-hexyl, n-heptyl and n-octyl-1-sulfonate.

[0061] Using such an IPR can make it difficult to detect peptides by mass spectrometry. Solutions are either to adapt the IPR concentrations used or to choose a more appropriate IPR.

[0062] In the context of the invention, the term “peptide elution” means the step of liquid chromatography where the peptides “exit” from the chromatography column with the mobile phase.

[0063] A "short sequence peptide" means a peptide consisting of less than 7 amino acid residues, in particular peptides consisting of 5 or 6 amino acids.

[0064] The term "peptide specific for an extended-spectrum beta-lactamase (ESBL)" means a peptide obtained by the hydrolysis of bacterial proteins of the beta-lactamase type, as defined in the introduction, and in particular ESBL enzymes of the TEM and SHV type, and indicating the "ESBL" character of the enzyme. This particularly concerns peptides having the following sequences:

[0065] - IHYSQNDLVK (SEQ ID NO. 4),

[0066] - LDHWEPELNEAIPNDER (SEQ ID NO. 5),

[0067] - LDSWEPELNEAIPNDER (SEQ ID NO. 6),

[0068] - SGASER (SEQ ID NO. 7),

[0069] - SGAGK (SEQ ID NO. 8),

[0070] - SGTGK (SEQ ID NO. 9),

[0071] - TGASER (SEQ ID NO. 25) and

[0072] - TGASK (SEQ ID NO. 26).

[0073] Scientific elements underlying the invention

[0074] The invention is based on low-resolution LC-MS / MS analysis of peptides derived from the enzymatic digestion of proteins from bacterial strains to detect the extended-spectrum β-lactamase (ESBL) type activities of TEM and SHV type enzymes expressed by enterobacteria.

[0075] An ion-pairing agent is used to increase the retention of short-sequence peptides on the stationary phase of chromatography, and thus to delay their elution with the mobile phase. The detection of these short-sequence peptides is indeed necessary to characterize bacterial strains exhibiting ESBL enzymatic activity. The articles by (Guo et al., 1987) and (Shibue et al., 2005) describe the effects of so-called "ion-pairing reagent" (IPR) compounds and their concentration on peptide retention, in the context of a reverse-phase liquid chromatography separation. In these peptide separation methods, the IPR is present in the mobile phase throughout the chromatographic separation. The effects of the presence of this IPR on subsequent analysis of the peptides by mass spectrometry are not addressed.

[0076] The article by (Gussakovsky et al., 2020) relates to the application of this method to a group of proteomic data comprising more than 12,000 peptides, analyzed by RPLC- MS / MS (Reverse Phase Liquid Chromatography - MS / MS spectrometry). The addition of 0.005% heptafluorobutyric acid (HFBA) in the mobile phase consisting of water and acetonitrile, acidified with 0.1% formic acid, increases the retention of peptides, up to 10% in acetonitrile for short hydrophilic sequence peptides. However, the ionic suppression due to the presence of HFBA results in a clear loss of sensitivity of the peptide signal (on average the signal reduction is 3.75 for the complete population of peptides).

[0077] The article (Sadjadi et al., 2017) concerns the separation of compounds such as aminoglycosides, catecholamines and paraquat, but does not provide guidance on the separation of peptides. This article shows that the retention time of the compounds to be separated is increased by adding an ion-pairing agent directly into the sample, rather than into the chromatography mobile phase. In this article, different IPRs were tested, including n-heptyl-1-sulfonate, added in concentrations ranging from 5 to 75 mM.

[0078] The use of ion-pairing agents has already been proposed to delay the elution of short sequence and / or hydrophilic peptides. However, this is the first time that this technology has been applied to the detection of peptides derived from ESBL proteins. Indeed, it was not foreseeable that the hydrolysis of ESBL proteins would generate so many peptides undetectable by a "conventional" use of targeted mass spectrometry, as described in patent EP 2 699 692.

[0079] Furthermore, the method of the invention comprises an addition of IPR in the chromatography column or in the sample, and not in the mobile phase of the chromatography, as described in previous studies relating to the detection of peptides.

[0080] Step (a) of the process of the invention: lysis of the bacteria and hydrolysis of the bacterial proteins

[0081] The bacteria present in the biological sample can be isolated by centrifugation. It is understood that this sample subjected to the method of the invention is a sample identified as “positive” during a prior step, that is to say containing within it bacteria whose possible resistance to antibiotics is unknown.

[0082] Different methods of bacterial cell lysis can be used. Examples include thermal methods (temperature above 100°C for 10 minutes, or freezing in liquid nitrogen), enzymatic methods (action of lysozyme or lyticase) or mechanical methods (high pressure, grinding or sonication).

[0083] According to a preferred implementation of the method of the invention, the lysis of the bacteria is carried out by sonication.

[0084] The hydrolysis of bacterial proteins into peptides is generally carried out by one of two processes: a) By the action of chemical agents such as hydroxyl radicals which induce random cleavages at the level of peptide bonds, or b) By the action of proteolytic enzymes (proteases) which have a hydrolysis action on protein bonds.

[0085] According to a preferred implementation of the method, the hydrolysis of bacterial proteins is an enzymatic hydrolysis.

[0086] Enzymatic digestion is carried out by enzymes called peptidases (or proteases) which hydrolyze the peptide bonds of proteins. Two classes of peptidases exist and are defined according to the location of the cut in the peptide chain: exopeptidases and endopeptidases.

[0087] Exopeptidases cleave the peptide chain between the terminal amino acid of the peptide chain and the rest of the chain. These enzymes are divided into two subclasses called aminopeptidases and carboxypeptidases depending on whether hydrolysis releases the N-terminal or C-terminal amino acid.

[0088] Endopeptidases can hydrolyze peptide bonds within the chain. Depending on their specificity, these enzymes can cleave bonds formed with certain amino acids. Among the best-known endopeptidases, trypsin allows the hydrolysis of peptide bonds at a carboxyl group engaged with a lysine or an arginine.

[0089] Among the commonly used proteases, we can cite pepsin which hydrolyzes peptide bonds preferentially before aromatic amino acids (Tyrosine, Tryptophan and Phenylalanine), endoproteinase GluC which cuts the peptide bond at the glutamate residues, or trypsin which cleaves proteins on the C-terminal side of the amino acids lysine and arginine.

[0090] According to a preferred implementation of the method of the invention, trypsin is used as an enzyme for the step of hydrolysis of bacterial proteins. Trypsin is in fact preferred for the specific nature of its activity, the adapted size of the peptides that it generates, and the nature of the “tryptic” peptides which have on the C-terminal side an amino acid which can be positively charged (lysine or arginine), thus facilitating analysis by mass spectrometry (analysis of charged molecules).

[0091] This step (a) also includes a centrifugation step to separate the mixture of peptides from the cellular debris; this step is well known to those skilled in the art and does not require further comment.

[0092] This step (a) may also comprise a step of adding an acid to the medium, intended to neutralize the enzyme carrying out the hydrolysis of the proteins after a sufficient incubation time; this is also well known to those skilled in the art.

[0093] Step (b): Analysis of the peptide mixture by targeted mass spectrometry coupled with reversed-phase chromatography, carried out in the presence of an ion-pairing agent

[0094] The present invention is based on a so-called “bottom-up” analysis technique as defined above.

[0095] According to one implementation of the invention, the mass spectrometry analysis is targeted during the analysis (SRM / MRM, MRM3, PRM type mode) or after the analysis (DIA / SWATH type mode).

[0096] Mass spectrometry is a physical analysis technology used to detect and identify molecules of interest. It is also known as single reaction monitoring, multiple reaction monitoring, or parallel reaction monitoring. Its principle lies in the gas-phase separation of charged molecules (ions) based on their mass-to-charge ratio (m / z).

[0097] Mass spectrometers comprise: i) an ionization source (called "electrospray") intended to ionize the molecules to be analyzed, i.e. to confer a positive or negative charge to these molecules; ii) a mass analyzer intended to separate the ionized molecules 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 below.

[0098] The ionization step required for the implementation of mass spectrometry can be implemented by any method known to those skilled in the art. The ionization source makes it possible to bring the molecules to be measured in a gaseous and ionized state. An ionization source can be used either in positive mode to study positive ions, or in negative mode to study negative ions. Several types of sources exist and will be used depending on the desired result and the molecules analyzed. It should be noted that both positive and negative modes cannot be used at the same time: thus, in example 4, only HFBA is monitored in negative mode, the peptides cannot be monitored during the same analysis because they must be monitored in positive mode.

[0099] The mass analyzer in which the step of separating the ionized markers according to their mass / charge ratio (m / z) is implemented is any mass analyzer known to those skilled in the art. Examples include low-resolution analyzers, such as quadrupole or quadrupole (Q), 3D ion trap (IT) or linear ion trap (LIT), also called ion trap, and high-resolution analyzers, which can measure the exact mass of the analytes and which use, in particular, the magnetic sector coupled to an electrical sector, the time of flight (TOF), and the orbitrap.

[0100] The separation of molecular ions based on their m / z ratio can be performed once (single mass spectrometry or MS), or several successive MS separations can be carried out. When two successive MS separations are performed, the analysis is called MS / MS or MS2. When three successive MS separations are performed, the analysis is called MS / MS / MS or MS3.

[0101] Targeted mass spectrometry is a variant in which the molecules of interest sought by this analysis technique are previously known, and the analysis is used to identify their presence or absence in a sample.

[0102] Targeted approaches (Multiple Reaction Monitoring, MRM; Parallel Reaction Monitoring, PRM; Multiple Reaction Monitoring-High Resolution, MRM-HR; Multiple Reaction Monitoring cubed, MRM3, DIA / SWATH) consist of the selection by a first analyzer of a precise mass corresponding to the peptide of interest which is then fragmented in a collision cell. The generated fragments are then monitored by a third analyzer and their signal / intensity is measured.

[0103] The principle of SRM mode, or MRM mode, is to specifically select a precursor ion, fragment it, and then specifically select one of its fragment ions. For such applications, triple quadrupole devices or triple quadrupole hybrids with ion traps are generally used.

[0104] The DIA / SWATH analysis mode consists of recording fragmentation spectra of contiguous precursor ion selection windows, most often overlapping by 1 mass-to-charge ratio (m / z) unit, the windows being characterized by a fixed or variable width in m / z, or by using a sliding window of fixed width in m / z, while ensuring that the total cycle time to cover all of these windows allows each chromatographic peak to be sampled by at least 5 points. In the case of chromatographic couplings, the fragment signal is measured as a function of time to be represented in the form of a chromatogram, the appearance of concomitant chromatographic peaks (simultaneous detection of fragments) therefore being evidence of the presence of the peptide in the sample.

[0105] These approaches require prior work of selection and validation of the targeted peptides to ensure their sequence / mass uniqueness (or mass / charge ratio, m / z) and their detectability.

[0106] According to one implementation of the invention, the mass spectrometry analysis is targeted during the analysis (SRM / MRM, MRM3, PRM type mode).

[0107] According to another implementation of the invention, the mass spectrometry analysis is targeted after the analysis (DIA / SWATH type mode).

[0108] More preferably, the targeted mass spectrometry analysis is carried out in MRM or MRM3 mode, and most preferably is carried out in MRM3 mode.

[0109] Targeted mass spectrometry is coupled with a reversed-phase liquid chromatography step. Reversed-phase chromatography is a widely used technique for separating peptides. The mobile phases used are volatile, which facilitates the concentration / lyophilization steps. The most commonly used solvents are water and acetonitrile.

[0110] During this chromatographic separation step, the peptides are "eluted," meaning they exit the chromatography column (stationary phase) with the mobile phase (liquid) at different times, depending on their charge and mass. The addition of an ion-pairing agent can delay this elution, as the peptides remain paired with the stationary phase for a little longer.

[0111] In the method of the invention, in order to counteract ionic suppression as much as possible and thus limit the loss of signal of the compounds studied, the ion pairing agent (IPR) is injected upstream of the analysis, i.e. added to the chromatography column, and not directly into the mobile phase. This agent can also be added directly into the peptide mixture, after enzymatic digestion.

[0112] We therefore distinguish two implementations of the method of the invention:

[0113] 1) The IPR is injected directly into the chromatography column, using an injector integrated into the system; it is therefore “added into the chromatography column”; or

[0114] 2) The IPR is added to the peptide mixture, which can also be referred to as the “sample”.

[0115] This technique is particularly suitable for subsequent analysis of compounds by mass spectrometry, as the IPR is completely eluted from the column during the washing step and therefore does not interfere with the subsequent step of compound analysis by mass spectrometry. In addition, this limits the progressive fouling of the mass spectrometer.

[0116] Figure 4 represents the elution over time of an IPR injected upstream of the analysis in the chromatography column: this is detectable by mass spectrometry in negative mode only during part of the analysis and not over its entire duration, unlike the case where the IPR would be added in the mobile phase.

[0117] Figure 3 highlights the impact of co-elution of IPR with peptides on the signal intensity measured by mass spectrometry. When the amount of IPR injected is too large, its elution zone coincides with that of the compounds of interest and this leads to a drastic drop in the signal relative to the peptides.

[0118] The preferred ion-pairing agent is heptafluorobutyric acid (HFBA), but any other compound capable of forming ion pairs can also be used. The acid concentration of the mobile phase was optimized to find the best possible compromise between retention gain and signal suppression.

[0119] IPR is present in a sufficient quantity to slow the elution of short sequence peptides, consisting of less than 7 amino acids. The person skilled in the art will know how to adapt the concentration of IPR according to its nature, the solvents used and the types of peptides to be retained.

[0120] The solvent used in reversed-phase chromatography is notably an aqueous solution of acetonitrile (H2O / ACN 98 / 2; v / v) acidified with 2% formic acid.

[0121] According to a particular implementation of the invention, heptafluorobutyric acid is used at a concentration of at least 0.4%, in an aqueous solution. The concentration of heptafluorobutyric acid can also be between 0.4% and 0.6%, or be equal to 0.4%, 0.5%, or 0.6%.

[0122] The IPR is either injected into the chromatography column upstream of the separation of the peptides on said column, or added to the mixture of peptides before its deposition on the chromatography column.

[0123] Peptides detected in the method according to the invention

[0124] According to one implementation of the invention, the extended-spectrum beta-lactamase (ESBL) whose presence in a sample is detected using the method of the invention is a TEM or SHV type enzyme.

[0125] As indicated above, the method of the invention is particularly suitable for detecting peptides of short sequence (less than 7 amino acids).

[0126] As mentioned, mass spectrometry (i.e., mass spectrometry analysis) is targeted, meaning that the presence of particular peptides is sought, these peptides being part of a predefined list. These are also referred to as "targeted peptides."

[0127] According to a particular implementation, the mass spectrometry is targeted for the detection of peptides consisting of 5 to 20 amino acids. According to a particular implementation of the invention, the mass spectrometry is targeted for the detection of peptides chosen from the group of 34 peptides having the following sequences: SEQ ID NO. 1 to SEQ ID NO. 34, as listed in Table 1 below.

[0128] According to a particular implementation of the method, mass spectrometry is targeted for the detection of peptides having a short sequence, i.e. consisting of less than 7 amino acids, in particular peptides consisting of 5 to 6 amino acids, and in particular peptides having the following sequences:

[0129] - SGASER (SEQ ID NO. 7)

[0130] - SGAGK (SEQ ID NO. 8)

[0131] - SGTGK (SEQ ID NO. 9)

[0132] - SGAGER (SEQ ID NO. 21) - TGASER (SEQ ID NO. 25)

[0133] - TGASK (SEQ ID NO. 26) and

[0134] - TGAGER (SEQ ID NO. 33)

[0135] According to another particular implementation of the method, the targeted peptides are in particular those indicative of an ESBL activity of the enzyme, and in particular the peptides having the following sequences: - IHYSQNDLVK (SEQ ID NO. 4),

[0136] - LDHWEPELNEAIPNDER (SEQ ID NO. 5),

[0137] - LDSWEPELNEAIPNDER (SEQ ID NO. 6),

[0138] - SGASER (SEQ ID NO. 7),

[0139] - SGAGK (SEQ ID NO. 8),

[0140] - SGTGK (SEQ ID NO. 9),

[0141] - TGASER (SEQ ID NO. 25) and

[0142] - TGASK (SEQ ID NO. 26).

[0143] Finally, according to another implementation of the method, the targeted peptides are particularly those having a short sequence and being indicative of an ESBL activity of the enzyme, and in particular the peptides having the following sequences:

[0144] - SGASER (SEQ ID NO. 7),

[0145] - SGAGK (SEQ ID NO. 8),

[0146] - SGTGK (SEQ ID NO. 9),

[0147] - TGASER (SEQ ID NO. 25) and

[0148] - TGASK (SEQ ID NO. 26).

[0149] Biological sample tested

[0150] According to a particular implementation of the invention, the enterobacterium present in the biological sample is of the species Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis or Enterobacter cloacae.

[0151] The sample on which the method of the invention is implemented is any sample likely to contain a target bacterial strain. This biological sample may be of human or animal origin.

[0152] According to one implementation of the invention, the biological sample is chosen from:

[0153] - A biological fluid such as blood, serum, plasma, urine, cerebrospinal fluid or tears,

[0154] - A bacterial culture such as a blood culture, an agar colony or a culture broth,

[0155] - A food sample, such as for example a sample of dairy product, meat, fish, egg, fruit, vegetable or a beverage, and

[0156] - any other type of biological sample, such as a water sample or a sample taken from a surface.

[0157] This will preferably be a biological fluid, in particular a blood sample (blood, serum, plasma).

[0158] This will more specifically be a positive blood culture. A "blood culture" means a blood sample taken from a patient and then incubated under suitable conditions to allow proliferation of any bacteria present in the sample. This blood culture may be carried out in blood culture bottles such as those sold in the Bact / Àlert range distributed by BioMérieux, or those in the Bactec range distributed by Becton Dickinson.

[0159] Kit

[0160] The present invention also relates to a kit for its use in implementing the method as described above, comprising reagents for implementing the method, in particular:

[0161] - an enzyme that allows the hydrolysis of proteins, such as trypsin, and

[0162] - an ion-pairing agent.

[0163] The ion-pairing agent may in particular be chosen from formic acid, perfluorinated acids such as trifluoroacetic acid (TFA), pentafluoropropionic acid (PFPA) and heptafluorobutyric acid (HFBA), as well as from n-butyl, n-pentyl, n-hexyl, n-heptyl and n-octyl-1-sulfonate compounds.

[0164] Preferably, it will be heptafluorobutyric acid.

[0165] Optionally, such a kit will also include preparation and / or use instructions, in particular a notice specifying the conditions for implementing the process.

[0166] EXAMPLES

[0167] The examples presented below are intended to illustrate the method according to the invention, but do not in any way limit the subject of the invention.

[0168] Example 1. Identification of peptides specific to TEM and SHV type ESBL enzymes

[0169] In order to determine the peptides to be followed to cover the maximum possible number of variants of the TEM and SHV enzymes, a preliminary study was carried out to identify peptides specific to these two enzymes, as well as peptides specific to the ESBL character. This study was carried out using the BLDB database.

[0170] Thus, the specific peptides of TEM-type enzymes, found in more than 95% of the variants, are SALPAGWFIADK (SEQ ID NO. 20), VDAGQEQLGR (SEQ ID NO. 22) and VGYIELDLNSGK (SEQ ID NO. 23). The peptide VAGPLLR (SEQ ID NO. 2) is also found in almost all TEM variants (99%), but cannot be followed by mass spectrometry as a specific marker of TEM. Indeed, the SHV protein has in its sequence the very similar peptide VAGPLIR (SEQ ID NO. 3) which is of identical mass.

[0171] SHV-specific peptides, found in more than 92% of the variants, are DTTTPASMAATLR (SEQ ID NO. 27), GIVALLGPNNK (SEQ ID NO. 28), QIGDNVTR (SEQ ID NO. 30) and QLLQWMVDDR (SEQ ID NO. 31). The peptides VAGPLIR (SEQ ID NO. 3) and FPMMSTFK (SEQ ID NO. 1) are also found in almost all the variants (>95%), but they cannot be followed to specifically characterize SHV because we find VAGPLLR (SEQ ID NO. 2, mass identical to the peptide of sequence SEQ ID NO. 3) and FPMMSTFK (SEQ ID NO.

[0172] 1) in the peptide sequence of the TEM-type enzyme. The study of the different possible mutations of TEM shows that the specific peptides indicating an ESBL activity are mainly: IHYSQNDLVK (SEQ ID NO. 4), LDSWEPELNEAIPNDER (SEQ ID NO. 6), LDHWEPELNEAIPNDER (SEQ ID NO. 5), SGASER (SEQ ID NO. 7), SGTGK (SEQ ID NO. 9), SGAGK (SEQ ID NO. 8).

[0173] Similarly, peptides specific for ESBL activity in SHV are TGASER (SEQ ID NO. 25) and TGASK (SEQ ID NO. 26).

[0174] By analyzing these peptides by LC-MS / MS, it is possible to identify all the most common TEM and SHV variants found in France in hospitals. It is also possible to identify the majority of existing ESBL variants available on the BLDB site: approximately 90% of the variants in TEM and 75% in SHV, knowing that these figures are probably underestimated since the database is incomplete for the less frequent variants.

[0175] The most frequently found TEM variants in France are shown in TABLE 2. The parts of the protein sequence in bold in the table correspond to the peptides to follow to know the resistance profile of the variant studied. The SHV variants most frequently found in France are shown in TABLE 3. The parts of the protein sequence in bold in the table correspond to the peptides to follow to determine the resistance profile of the variant studied.

[0176] Example 2. Adjusting parameters to optimize peptide retention time

[0177] Several concentrations of heptafluorobutyric acid (HFBA) were tested as part of the development of the process according to the invention.

[0178] In practice, 5 pL of HFBA solution was injected into the chromatography column before injecting 5 L of peptide mixture sample; this peptide mixture is included in an acidic aqueous solution comprising 2% formic acid (FA), this acidification of the medium making it possible to stop the enzymatic digestion by trypsin.

[0179] The following percentages were tested:

[0180] - 0.05% HFBA (3.8 mM)

[0181] - 0.1% HFBA (7.6 mM)

[0182] - 0.2% HFBA (16.2 mM)

[0183] - 0.3% HFBA (23.8 mM)

[0184] - 0.4% HFBA (31.4 mM)

[0185] - 0.5% HFBA (38 mM)

[0186] The results obtained for 7 peptides: SGASER (SEQ ID NO.7), SGAGER (SEQ ID NO.21), TGASER (SEQ ID NO.25), TGAGER (SEQ ID NO.33), SGAGK (SEQ ID NO.8), TGASK (SEQ ID NO.26), and SGTGK (SEQ ID NO.9), are presented in Figure 2.

[0187] It clearly appears that the retention of peptides consisting of 5 or 6 amino acids is satisfactory from a percentage of 0.4% (31 mM) of HFBA.

[0188] Example 3. Identification of resistance profiles (ESBL or penicillinase) of enterobacteriaceae strains expressing a TEM or SHV type enzyme

[0189] The bacterial samples that were analyzed by LC-MS / MS are reported in Table 4 below. These samples are agar plates (bacterial culture), but the analysis presented can also be performed with blood cultures. All these samples were subjected to enzymatic digestion. The protocol used is as follows. The first 3 steps are optional and are only performed if sample pre-concentration is necessary.

[0190] 1) Centrifugation of 1 mL of bacterial culture at 10,000 rpm for 5 minutes

[0191] 2) Removal of the supernatant, conservation of only the bacterial pellet

[0192] 3) Resumption of the sample in 200 pL of water (MS grade)

[0193] 4) Adding glass beads (approximately 1 / 3 of the final volume)

[0194] 5) Addition of 50 pL of trypsin solution (1 mg / mL in 150 mmol ammonium bicarbonate)

[0195] 6) Ultrasound treatment (Bioruptor® from Diagenode), 10 cycles of 30 seconds of ultrasound and 30 seconds of rest at 50°C

[0196] 7) Stop digestion with 5 pL of formic acid

[0197] 8) Centrifugation of the sample at WOOOrpm for 5 minutes

[0198] 9) Analysis of the supernatant (peptide mixture) by LC-MS / MS.

[0199] The chromatographic conditions for the analyses are as follows:

[0200] • Chromatographic chain: Agilent 1290 Infinity from Agilent Technologies

[0201] • Column: Waters XBridge® Peptide BEH C18, 2.1x100mm, 3.5mm particle size, 130Â

[0202] • Oven: PerkinElmer Series 200 Peltier Column

[0203] • Solvent A: Water (H2O) + 0.1% formic acid

[0204] • Solvent B: Acetonitrile (ACN) + 0.1% formic acid

[0205] • Mobile phase flow rate: 300 pL / min

[0206] • Oven temperature: 60°C

[0207] • Injection volumes: 5 pL of HFBA solution (0.4% in H2O), then 5 pL of sample

[0208] The mobile phase gradient used is shown in TABLE 5 below. The program used for injection is as follows:

[0209] • Take 5 pL from the vial containing the HFBA solution

[0210] • Inject

[0211] • Take 5 pL from the vial containing the sample to be analyzed

[0212] • Inject

[0213] If the system used does not allow for an injection program, then it is also possible to use two chromatographic methods in succession to analyze the samples. The first method would be isocratic at 98% mobile phase A and lasting a few seconds to inject the acid at the top of the column. The second method would allow the sample to be injected and analyzed with the classic chromatographic conditions described above and the mobile phase gradient shown in TABLE 5.

[0214] If the implementation of these two analysis methods is a hindrance for the user, it is also possible to add the HFBA directly to the sample and carry out a simple injection, which would add a step to the sample preparation.

[0215] Liquid chromatography is coupled to a Sciex 6500+ mass spectrometer. Peptides resulting from the enzymatic digestion of bacterial strains are analyzed by mass spectrometry in MRM mode (positive electrospray ionization). Peptides specific to TEM and SHV type enzymes, as well as ribosomal peptides found in enterobacteria were monitored. The 42 peptides monitored in the method are described in TABLE 6 below.

[0216] 34 peptides are derived from a TEM and / or SHV type enzyme; among these, 8 are indicative of the ESBL activity of the enzyme considered; and 8 peptides are ribosomal peptides detected for the purpose of normalization / quantification.

[0217] Table 6.

[0218] The parameters used to detect the transitions cited in TABLE 6 are:

[0219] • The retention times of the different peptides

[0220] • The m / z (mass to charge) ratios of the precursor and fragment ions • The collision energy (CE - Collision Energy)

[0221] • The dissociation or orifice potential (DP - Declustering Potential)

[0222] • The collision cell exit potential (CXP - Cell eXit Potential)

[0223] These parameters are described in TABLE 7 for each of the transitions.

[0224] Table 7.

[0225] The other mass parameters used for the analysis of all samples are:

[0226] Scheduled AARM Polarity: positive

[0227] Ionization source: Turbo Spray

[0228] MRM detection window: 60 seconds

[0229] - Cycle time: 1.32 seconds

[0230] Input Potential (EP): 10 V

[0231] Curtain gas (CUR - Curtain Gas): 50 psi

[0232] Collision Gas (CAD): high

[0233] - Cone voltage (IS - lonSpray Voltage): 5500 V

[0234] - Temperature (TEM): 550° C

[0235] Auxiliary Gas 1 (Ion Source Gas 1): 70 psi

[0236] Auxiliary Gas 2 (Ion Source Gas 2): 60 psi

[0237] Resolution Q1: unit

[0238] Resolution Q3: unit

[0239] Pause between mass changes: 5.007 ms

[0240] To avoid fouling of the device caused by dead volume, the beginning of the analysis is not analyzed by mass spectrometry. Ionization of the compounds begins after 1.2 minutes.

[0241] The detection of each peptide is validated if the studied transitions are aligned, if the intensity ratios between the transitions are consistent with those of synthetic peptides identical to those analyzed, and finally if the detected intensity threshold is equivalent to at least three times the noise intensity for the studied transition. The results obtained are described in TABLE 8A, TABLE 8B, TABLE 8C. The “-” corresponds to the absence of detection while “yes” indicates the presence of the peptide. When the presence of the peptide implies an ESBL-type enzymatic activity, the “yes” is in bold.

[0242] Table 8A.

[0243]

[0244] Table 8B.

[0245]

[0246] Table 8C.

[0247] All samples tested contain at least one peptide specific to a TEM or SHV type enzyme. The bacterial strains tested are therefore at least resistant to the action of penicillins and first-generation cephalosporins. Samples Ech 1, Ech 2, Ech 4, Ech 6, Ech 9, Ech 10, Ech 16, Ech 17, Ech 18, Ech 19, Ech 25, Ech 26, Ech 27, Ech 28, Ech 29, Ech 30 contain at least one peptide specific to the ESBL character of these enzymes. These bacterial strains are therefore resistant to penicillins, first- to fourth-generation cephalosporins, and monobactams. At least one peptide among SGAGER, SGASER, SGAGK, SGTGK, TGAGER, TGASER and TGASK was detected in samples Ech 1, Ech 3, Ech 4, Ech 6, Ech 7, Ech 9, Ech 1 1, Ech 13, Ech 15, Ech 16, Ech 17, Ech 18, Ech 19, Ech 20, Ech 25, Ech 27, Ech 28, Ech 29 and Ech 30. Figure 1 shows a chromatogram which illustrates the detection of the SGAGK peptide (SEQ ID NO. 8) in the Ech 27 sample.

[0248] Among the samples with ESBL-type enzymatic activity cited above, samples Ech 6 and Ech 9 had their resistance profile correctly characterized thanks to the identification of 6-amino acid peptides, respectively TGASER (SEQ ID NO. 25) and SGASER (SEQ ID NO. 7), which are the only ESBL-specific peptides found in these samples.

[0249] The analysis method according to the invention, using HFBA to better retain certain peptides, therefore makes it possible to identify strains of enterobacteria with an ESBL character which could not have been detected under conventional chromatographic conditions (reverse phase and usual mobile phase).

[0250] Example 4. Adding HFBA directly to the chromatography column rather than to the mobile phase does not reduce the sensitivity of peptide signal detection.

[0251] Figures 3 and 4 illustrate the advantage of adding HFBA only upstream of the injection, i.e. directly onto the chromatography column.

[0252] Synthetic peptides with sequences TGASK, TGAGER, TGASER, SGAGER, and SGASER were used to test different conditions of use of HFBA, an ion-pairing agent.

[0253] Figure 3 shows the signal loss measured when HFBA is mixed with the chromatography mobile phase. In this case, HFBA co-elutes with the peptides of interest.

[0254] When detected by tandem mass spectrometry, the signal quantity of the peptides TGASK, TGAGER, TGASER, SGAGER, SGASER is divided by 5, or even by 8 for the peptide TGASK. For SGAGK, the signal is divided by 2, probably because its chromatographic peak only partially co-elutes with that of HFBA. Finally, for SGTGK, the latter does not co-elute under the conditions of the experiment with HFBA, so there is no suppression of ions from its signal.

[0255] Figure 3 highlights the impact of co-elution of IPR with peptides on the signal intensity measured by mass spectrometry. When the amount of IPR injected is too large, its elution zone coincides with that of the compounds of interest and this leads to a drastic drop in the signal relative to the peptides.

[0256] Figure 4 is a chromatogram obtained by monitoring, exclusively in negative mode, the HFBA signal. The quantity of HFBA injected upstream of the analysis is 30 mM.

[0257] The chromatographic method used is the same as that used for the peptide analysis above. This figure represents the elution over time of HFBA: the elution zone of HFBA is distributed only over a part of the chromatogram, which corresponds to the area located after the elution of the five- and six-amino acid peptides. Thus, it is detectable only during a part of the analysis, and not over its entire duration, unlike the case where the IPR is added directly to the mobile phase.

[0258] BIBLIOGRAPHICAL REFERENCES

[0259] EP 2 699 692

[0260] US 2017 / 0052198

[0261] Bush, K.; Jacoby, G. A. Updated Functional Classification of B-Lactamases. Antimicrob. Agents Chemother. 2010, 54 (3), 969-976. https: / / doi.org / 10.1128 / AAC.01009-09.

[0262] Oviano, M.; Fernandez, B.; Fernandez, A.; Barba, M. J.; Mourino, C.; Bou, G. Rapid Detection of Enterobacteriaceae Producing Extended Spectrum Beta -Lactamases Directly from Positive Blood Cultures by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry. Clin. Microbiol. Infect. 2014, 20 (11 ), 1146-1157. https: / / doi.org / 10.1111 / 1469-0691.12729.

[0263] Hou, T.-Y.; Chiang-Ni, C.; Teng, S.-H. Current Status of MALDI-TOF Mass Spectrometry in Clinical Microbiology. J. Food Drug Anal. 2019, 27 (2), 404-414. https: / / doi.Org / 10.1016 / j.jfda.2019.01.001.

[0264] Pupo, G.; Lan, R.; Reeves, P. Multiple Independent Origins of Shigella Clones of Escherichia Coli and Convergent Evolution of Many of Their Characteristics. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 10567-10572. https: / / doi.org / 10.1073 / pnas.180094797.

[0265] Shibue M., Mant C. T., Hodges R. S. Effect of anionic ion-pairing reagent concentration (1 - 60 mM) on reversed-phase liquid chromatography elution behaviour of peptides. J.

[0266] Chromatogr A.2005;1080:58 67

[0267] Guo D. C., Mant C. T., Hodges R. S. Effects of ion-pairing reagents on the prediction of peptide retention in reversed-phase high-performance liquid chromatography. J. ChromatogrA. 1987;386:205-22

[0268] Fleurbaaij F, Goessens W, van Leeuwen HC, Kraakman MEM, Bernards ST, Hensbergen PJ, Kuijper EJ. Direct detection of extended -spectrum beta- lactamases (CTX-M) from blood cultures by LC-MS / MS bottom-up proteomics. Eur J Clin Microbiol Infect Dis. 2017 Sep;36(9):1621 -1628.

[0269] Sadjadi, S.; Layne, J.; Preston, J. A New Twist to Ion-Pairing Chromatography: In-Sample Addition of Ion-Pairing Reagent. LCGC N. Am. 2017, 35, 824-831.

[0270] Gussakovsky D., Anderson G., Spicer V., Krokhin O. V. Peptide separation selectivity in proteomics LC-MS experiments: Comparison of formic and mixedformic / heptafluorobutyric acids ion-pairing modifiers. Journal of Separation Science 2020 ;43: 3830-3839

Claims

DEMANDS 1. A method for determining the resistance properties to beta-lactam antibiotics of an enterobacterial strain present in a biological sample, comprising the following steps: a) lysis of the bacteria and hydrolysis of the bacterial proteins, to obtain a mixture of peptides, b) analysis of this mixture of peptides by targeted mass spectrometry coupled to reversed-phase liquid chromatography, the detection of at least one peptide specific to an enzyme with extended-spectrum beta-lactamase (ESBL) activity being indicative of the resistance to beta-lactams of the enterobacterial strain present in said biological sample, said method being characterized in that the analytical step (b) is carried out in the presence of an ion-pairing agent (IPR) added to the chromatography column or to the mixture of peptides.

2. A method according to claim 1, characterized in that the IPR is present in sufficient quantity to slow down the elution of peptides of less than 7 amino acid residues.

3. A method according to any one of claims 1 to 2, characterized in that the mass spectrometry analysis is targeted during the analysis (SRM / MRM, MRM3, PRM type mode) or after the analysis (DIA / SWATH type mode).

4. A process according to any one of claims 1 to 3, characterized in that the ion-pairing agent is heptafluorobutyric acid.

5. A process according to claim 4, characterized in that heptafluorobutyric acid is present in an aqueous solution at a concentration of at least 0.4%.

6. A process according to any one of claims 1 to 5, characterized in that the hydrolysis of bacterial proteins in step (a) is an enzymatic hydrolysis, in particular is a hydrolysis by trypsin.

7. A method according to any one of claims 1 to 6, characterized in that mass spectrometry is targeted for the detection of peptides consisting of 5 to 20 amino acids.

8. A method according to any one of claims 1 to 7, characterized in that mass spectrometry is targeted for the detection of peptides selected from the group of 34 peptides having the following sequences: SEQ ID NO. 1 to SEQ ID NO.

34.

9. A method according to claim 8, characterized in that mass spectrometry is targeted for the detection of peptides selected from a group of 7 peptides having the following sequences: - SGASER (SEQ ID NO. 7) - SGAGK (SEQ ID NO. 8) - SGTGK (SEQ ID NO. 9) - SGAGER (SEQ ID NO. 21) - TGASER (SEQ ID NO. 25) - TGASK (SEQ ID NO. 26) - TGAGER (SEQ ID NO. 33).

10. A method according to any one of claims 1 to 9, characterized in that the enterobacterium is of the species Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis or Enterobacter cloacae.

11. A method according to any one of claims 1 to 10, characterized in that the biological sample is chosen from: - A biological fluid such as blood, serum, plasma, urine, cerebrospinal fluid, or tears, - A bacterial culture such as a blood culture, a colony on agar, or a culture broth, - A food sample, and - any other type of biological sample.

12. Kit for its use in carrying out the process according to any one of the claims 1 to 11, including trypsin and heptafluorobutyric acid.

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