Patent Grant
9506932
The present invention relates to the field of microbiology. More precisely, the invention relates to the detection of at least one mechanism of resistance to cephalosporins of at least one microorganism from a sample by using mass spectrometry.
Since Pasteur's discovery of microbes, microorganisms have been studied by microscopy and biochemical analyses. These conventional methods are often long and tedious, and analytical alternatives were sought very early on. This is why the analysis of bacteria by mass spectrometry was initiated from 1975 by J. Anhalt and C. Fenselau [1].
This preliminary work was followed by the study of fatty acids from the wall of the microorganisms using gas chromatography combined with mass spectrometry (GC-MS) [2]. This method was popularised under the English term FAME, standing for Fatty Acid Methyl Ester. It currently constitutes a reference method for taxonomic studies. However, its use remains limited to certain specialised laboratories dealing with the treatment of the sample by saponification, hydrolysis and derivation.
In 1996, the works by M. Claydon et al. [3] as well as by T. Krishnamurthy and P. Ross [4] demonstrated the possibility of identifying different bacterial species with a MALDI-TOF mass spectrometer (English acronym for Matrix Assisted Laser Desorption Ionization—Time Of Flight). The analysis combines the acquisition of a mass spectrum and the interpretation of expert software. It is extremely simple and can be carried out in a few minutes. However it has only been making it into medical analysis laboratories fairly recently [5]. Its clinical use is currently limited to the identification of bacteria and yeast species. It is not routinely used to identify resistances to antimicrobials.
Yet the identification of resistances to antimicrobials such as antibiotics is an essential element in ensuring optimal patient care.
Other mass spectrometry methods, particularly in tandem, have been proposed to meet these needs. By way of example, it is possible to cite the work of C. Fenselau et al. for identifying β-Lactamase with a quadripole-TOF (Q-TOF) [6].
However these research results are not applicable to routine clinical use. They were obtained with research instruments requiring highly qualified personnel. The analysis times, often greater than one hour per sample, are incompatible with the workload of a microbiological analysis laboratory.
More recently, S. Hofstadler et al. [7] proposed a method combining a microbial genome amplification by PCR to a detection of the PCR products by electrospray-TOF (ESI-TOF). This method is now fully automated [8]. However, it requires a PCR amplification with the flaws inherent in molecular biology, namely extraction yield, cost of the probes, etc.
In this context, the objective of the present invention is to propose a method of detecting mechanisms of resistance to cephalosporins which makes it possible to overcome the disadvantages of the prior art methods, namely providing an inexpensive method, without reagents specific to each species, particularly compared to molecular biology methods, which gives a result in a short amount of time, less than one hour, and which can be used in routine clinical work, without requiring highly qualified personnel.
To this end, the invention proposes a new method of detecting, by mass spectrometry, at least one mechanism of resistance to at least one antimicrobial of at least one microorganism from a sample, characterised in that the antimicrobial is a cephalosporin and in that proteins and/or peptides are detected as markers of said mechanism of resistance to at least one cephalosporin-class antibiotic.
Advantageously, markers of resistance to several different antimicrobials can be detected simultaneously.
As indicated in application PCT/FR2010/052181, markers of type and/or virulence of said microorganisms can be detected in the same way by mass spectrometry prior to or at the same time as the detection of the resistance mechanism markers.
Markers of resistance to at least one cephalosporin-class antimicrobial is understood to mean molecules of protein origin which are characteristic of said properties. Cephalosporins are antibiotics belonging to the beta-lactam family. They are usually classified in several subclasses:
Determination of the resistance to at least one antimicrobial is understood to mean determining the susceptibility of a microorganism to being destroyed by an antimicrobial. The proteins involved in the resistance mechanisms will differ depending on the family and the species.
The nomenclature of the beta-lactamases, beta-lactam-resistant bacterial enzymes, is not standardised. They are either classified in four molecular classes (A to D) on the basis of their primary structure, or in functional groups on the basis of the target substrates and their resistance to inhibitors (for an overview, see [9] Bush and Jacoby, Antimicrobial Agents and Chemotherapy, 2010; 54 (3): 969-976). For molecular classification, sequencing techniques have made more precise classification possible: for example, 183 variants of the TEM protein have been described (labelled TEM-i, with i being between 1 and 183). For the functional classification, Bush and Jacoby (supra) have proposed new functional subgroups:
The method of the invention can be employed to detect mechanisms of resistance to cephalosporins in bacteria. Thus, for example, as bacteria in which it is possible to seek a mechanism of resistance to cephalosporins according to the method of the invention, non-exhaustive mention may be made of:
It should further be noted that the strains known to be resistant to carbapenems are also resistant to cephalosporins and to penicillins. Therefore, a method of detecting a mechanism of resistance to carbapenems also makes it possible to detect a mechanism of resistance to cephalosporins and to penicillins.
The sample on which the method of the invention can be employed is any sample susceptible of containing a target microorganism. The sample can be of biological origin, either animal, vegetable or human. In this case it may correspond to a specimen of biological fluid (whole blood, serum, plasma, urine, cerebrospinal fluid, organic secretion, for example), a tissue specimen or isolated cells. This specimen can be used such as it is insofar as the markers of mechanisms of bacterial resistance to beta-lactams are available in the sample tested, or it can, prior to the analysis, undergo preparation by enrichment, extraction, concentration, purification, culturing, in accordance with methods known to the person skilled in the art.
The sample can be of industrial origin, or, according to a non-exhaustive list, can be an air specimen, a water specimen, a surface specimen, a part or a manufactured product, or a food product. Amongst the food samples, non-exhaustive mention can be made of a sample of a dairy product (yogurts, cheeses), of meat, of fish, of egg, of fruit, of vegetable, of water, of a beverage (milk, fruit juice, soda, etc.). These food samples can also come from sauces or ready meals. Finally, a food sample can come from an animal feed, such as animal meals.
Upstream of the detection by mass spectrometry, the sample to be analysed is preferably pre-treated to produce peptides from the entirety of the proteins present in the sample to fragment these proteins into peptides, for example by digestion with a proteolytic enzyme (protease), or by the action of a chemical reagent. In fact, the cleaving of the protein can be performed by a physico-chemical treatment, by a biological treatment or by a combination of the two treatments. Amongst the useable treatments, mention can be made of treatment by hydroxyl radicals, in particular with H2O2. Treatment by hydroxyl radicals results in a cutting of the peptide bonds which takes place randomly on any of the protein's peptide bonds. The hydroxyl radical concentration determines the number of cleavages performed, and therefore the length of the peptide fragments obtained. Other chemical treatments can also be used such as, for example, cyanogen bromide (CNBr) treatment which specifically splits the peptide bonds at the carboxyl group of the methionyl residues. It is also possible to perform partial acid cleaving at the aspartyl residues by heating a solution of proteins in trifluoroacetic acid to 1000° C.
Treatment of the proteins by enzymatic digestion is nevertheless preferred over physico-chemical treatment because it preserves more of the structure of the protein, and is easier to control. “Enzymatic digestion” is understood to mean the single or combined action of one or more enzymes under appropriate reaction conditions. The enzymes carrying out the proteolysis, which are called proteases, cut the proteins at specific locations. Each protease generally recognises a sequence of amino acids within which it always makes the same cut. Certain proteases recognise a single amino acid or a sequence of two amino acids between which they perform a cleavage, whereas other proteases only recognise longer sequences. These proteases can be endoproteases or exoproteases. Amongst the known proteases, mention may be made of the following as described in WO20051098071:
Several proteases may be used simultaneously, if their modes of action are compatible, or they may be used successively. Within the framework of the invention, the digestion of the sample is preferably performed by the action of a protease enzyme, for example trypsin.
The generation of peptides using a chemical reagent or a protease can be obtained by means of a simple reaction in solution. It can also be performed with a microwave oven [10], or under pressure [11], or even with an ultrasound device [12]. In these three latter cases, the protocol will be much faster.
Amongst the peptides thus obtained, the peptides specific to the protein are referred to as proteotypic peptides. It is these which will be assayed by mass spectrometry. According to the invention, the markers of the mechanisms of bacterial resistance to cephalosporins are proteins from the bacterium in which the mechanisms of resistance to cephalosporins are to be sought. In particular, said proteins are digested into peptides, preferably by an enzyme, and more preferably by trypsin.
Similarly, the sample containing protein markers characterising mechanisms of bacterial resistance to cephalosporins can also be pre-treated for the purposes of purification. This purification pretreatment can be employed before or after the peptide production step as described above.
The sample purification pretreatment is widely known to the person skilled in the art and may in particular employ the techniques of centrifugation, filtration, electrophoresis or chromatography. These separating techniques can be used alone or in combination with one another to obtain a multidimensional separation. For example, multidimensional chromatography can be used by combining separation by ion exchange chromatography with reversed-phase chromatography, as described by T. Fortin et al. [13], or H. Keshishian et al. [14]. In these publications, the chromatography medium can be in a column or in a cartridge (solid-phase extraction).
The electrophoretic or chromatographic fraction (or the retention time in mono-dimensional or multidimensional chromatography) of the proteotypic peptides is characteristic of each peptide, and employing these techniques therefore makes it possible to select the proteotypic peptide or peptides to be assayed. Such a fractionation of the produced peptides makes it possible to increase the specificity of the subsequent assay by mass spectrometry.
An alternative to the electrophoresis or chromatography techniques for the fractionation of the peptides consists in specifically purifying the N-glycopeptides ([15] and patent application WO 2008/066629). However, such a purification only makes it possible to quantify the peptides which have undergone an N-glycosylation post-translational modification. Not all proteins are glycosylated though, which therefore limits its use.
The mass spectrometry to be employed in the method of the invention is widely known to the person skilled in the art as a powerful tool for analysing and detecting different types of molecules. Generally, any type of molecule able to be ionised can be detected according to its molecular mass with the aid of a mass spectrometer. According to the nature of the molecule to be detected, whether of protein or metabolic origin, certain mass spectrometry technologies can be more suitable. Nevertheless, whatever mass spectrometry method is used for the detection, this latter includes a step of ionising the target molecule into so-called molecular ions, in the present case a step of ionising the characterising markers, and a step of separating the molecular ions obtained according to their mass.
All mass spectrometers therefore comprise:
The ionisation step necessary for employing mass spectrometry can be performed via any method known to the person skilled in the art. The ionising source makes it possible to transform the molecules to be assayed into a gaseous and ionised state. An ionising source can be used either in positive mode to study the positive ions, or in negative mode to study the negative ions. Several types of sources exist and will be used depending on the result sought and the molecules analysed. In particular, mention may be made of:
In particular, ionisation can be employed as follows: the sample containing the target molecules is introduced into an ionisation source, where the molecules are ionised in gaseous state and thus transformed into molecular ions which correspond to the initial molecules. An electrospray ionisation (ESI) source makes it possible to ionise a molecule by making it pass from a liquid state into a gaseous state. The molecular ions obtained therefore correspond to the molecules present in liquid state, with, in positive mode, one, two, or even three or more additional protons and therefore carry one, two, or even three or more charges. For example, when the target molecule is a protein, an ionisation of the proteotypic peptides obtained after fractionation of the target protein, by means of an electrospray source functioning in positive mode, leads to polypeptide ions in gaseous state, with one, two, or even three or more additional protons and which therefore carry one, two, or even three or more charges, and makes it possible to move from a liquid state to a gaseous state [16]. This type of source is particularly well suited when the target molecules or proteotypic peptides obtained are separated beforehand by reversed-phase liquid chromatography. Nevertheless, the ionisation yield of the molecules present in the sample may vary depending on the concentration and the nature of the different species present. This phenomenon leads to a matrix effect well known to the person skilled in the art.
A MALDI ionisation source will allow ionisation of the molecules from a solid-state sample.
The mass analyser in which the step of separating the ionised markers according to their mass-to-charge ratio (m/z) is performed is any mass analyser known to the person skilled in the art. Mention can be made of low-resolution analysers, quadripole or quadrupole (Q), 3D ion trap (IT) or linear ion trap (LIT), also called ion trap, and high-resolution analysers which make it possible to measure the exact mass of the analytes and which in particular use the magnetic sector linked to an electric sector, the time of flight (TOF), Fourier transform ion cyclotron resonance (FT-ICR), orbitrap.
The separation of the molecular ions depending upon their m/z ratio can be employed just once (single mass spectrometry or MS), or several successive MS separations can be conducted. When two successive MS separations are carried out, the analysis is called MS/MS or MS2. When three successive MS separations are carried out, the analysis is called MS/MS/MS or MS3, and more generally, when n successive MS separations are carried out, the analysis is called MSn.
Amongst the techniques which employ several successive separations, SRM (Selected Reaction Monitoring) mode when detecting or assaying a single target molecule, or MRM (Multiple Reaction Monitoring) mode when detecting or assaying several target molecules are particular uses of MS2 separation. Similarly the MRM3 mode is a particular use of MS/MS/MS separation. This is referred to as targeted mass spectrometry.
In the case of a detection in single MS mode, it is the mass-to-charge ratio of the molecular ions obtained which is correlated to the target molecule to be detected.
In the case of detection in MS/MS mode, essentially two steps are added, compared to an MS assay, which are:
It is therefore the mass-to-charge ratio of the 1st generation fragment ions thus obtained which is correlated to the target molecule to be detected. First-generation fragment ion is understood to be an ion derived from the precursor ion, following a fragmentation step and of which the mass-to-charge ratio m/z is different from the precursor ion.
The (m/z)1 and (m/z)2 pairs are called transitions and are representative of the characteristic ions to be detected.
The choice of the characteristic ions which are detected to be correlated to the target molecule is made by the person skilled in the art in accordance with the standard methods. Their selection will advantageously lead to the most sensitive, specific and robust assays possible, in terms of reproducibility and reliability. In the methods developed for the selection of proteotypic peptides (m/z)1, and of the first-generation fragment (m/z)2, the choice is essentially based on the intensity of the response. For more details, it is possible to refer to V. Fusaro et al. [17]. Commercially available software, such as the MIDAS and MRM Pilot software from Applied Biosystems or MRMaid [18] can be used by the person skilled in the art to allow him to predict all the possible transition pairs. He can also make use of a database called PeptideAtlas constructed by F Desiere et al. [19] to compile all of the MRM transitions of peptides described by the scientific community. This database PeptideAtlas is freely available on the internet. For non-protein molecules, it is also possible to use databases, such as, for example, the one accessible through the Cliquid software from the company Applied Biosystems (United States of America).
An alternative approach to selecting the proteotypic peptides (m/z)1 and (m/z)2 consists in using MS/MS fragmentation spectra obtained during other work. This work can be, for example, the phases of biomarker discovery and identification by proteomic analysis. This approach was proposed by Thermo Scientific during user conferences [18]. It makes it possible to generate a list of candidate transitions from the peptides identified through testing by the SIEVE (Thermo Scientific) software. Certain criteria were detailed by J. Mead et al. [18] for the choice of the ions (m/z)1 and (m/z)2 and are detailed hereafter:
The fragmentation of the selected precursor ions is performed in a fragmentation cell such as the triple quadripole model [20], ion trap model [21], or time-of-flight (TOF) model [22], which also make it possible to separate ions. The fragmentation or fragmentations will be conventionally performed by collision with an inert gas such as argon or nitrogen, within an electrical field, by photo-excitation or photo-dissociation using an intense light source, collision with electrons or radical species, by applying a potential difference, for example in a time-of-flight tube, or by any other activation to mode. The characteristics of the electrical field determine the intensity and nature of the fragmentation. Thus, the electrical field applied in the presence of an inert gas, for example in a quadripole, determines the collision energy provided to the ions. This collision energy will be optimised, by the person skilled in the art, to increase the sensitivity of the transition to be assayed. By way of example, it is possible to vary the collision energy between 5 and 180 eV in q2 in an AB SCIEX QTRAP® 5500 mass spectrometer from the company Applied Biosystems (Foster City, United States of America). Similarly, the duration of the collision step and the excitation energy within, for example, an ion trap will be optimised by the person skilled in the art to lead to the most sensitive assay. By way of example, it is possible to vary this duration, called excitation time, between 0.010 and 50 ms and the excitation energy between 0 and 1 (arbitrary unit) in Q3 in an AB SCIEX QTRAP® 5500 mass spectrometer by the company Applied Biosystems.
Finally, the detection of the selected characteristic ions takes place in the conventional manner, particularly by means of a detector and a processing system. The detector collects the ions and produces an electrical signal whose intensity depends on the amount of ions collected. The signal obtained is then amplified such that it can be processed by computer. A computer data processing assembly makes it possible to transform the information received by the mass spectrum detector.
The principle of the SRM mode, or even of the MRM mode, is to specifically select a precursor ion, fragment it, and then specifically select one of its fragment ions. For such applications, triple quadripole or hybrid triple quadripole/ion trap devices are generally used.
In the case of a triple quadripole device (Q1q2Q3) used in MS2 mode, with a view to assaying or detecting a target protein, the first quadripole (Q1) makes it possible to filter the molecular ions corresponding to the proteotypic peptides characteristic of the protein to be assayed and obtained during an earlier digestion step, depending on their mass-to-charge ratio (m/z). Only the peptides having the mass-to-charge ratio of the proteotypic peptide sought, which ratio is called (m/z)1, are transmitted into the second quadripole (q2) and act as precursor ions for the subsequent fragmentation. The analyser q2 can fragment the peptides of mass-to-charge ratio (m/z)1 into first-generation fragment ions. Fragmentation is generally obtained through collision of the precursor peptides with an inert gas, such as nitrogen or argon in q2. The first-generation fragment ions are transmitted into a third quadripole (Q3) which filters the first-generation fragment ions depending on a specific mass-to-charge ratio, called (m/z)2. Only the first-generation fragment ions having the mass-to-charge ratio of a fragment characteristic of the sought proteotypic peptide (m/z)2 are transmitted into the detector in order to be detected, or even quantified.
This mode of operation exhibits a double selectivity, with regard to the selection of the precursor ion on the one hand, and the selection of the first-generation fragment ion on the other hand. Mass spectrometry in SRM or MRM mode is therefore advantageous for quantification.
When the mass spectrometry employed in the method according to invention is tandem mass spectrometry (MS2, MS3, MS4 or MS5), several mass analysers can be linked to one another. For example, a first analyser separates the ions, a collision cell makes it possible to fragment the ions, and a second analyser separates the fragment ions. Certain analysers, such as the ion traps or the FT-ICR, constitute several analysers in one and make it possible to fragment the ions and analyse the fragments directly.
According to preferred embodiments of the invention, the method of the invention comprises one or more of the following characteristics:
In addition to determining the resistance to an antibiotic, it is necessary to identify the microorganism or microorganisms present in the sample to be tested.
The methods of identifying microorganisms are widely known to the person skilled in the art, as described for example by Murray P. R. et al. in Manual of Clinical Microbiology, 2007, 9th edition, and especially in Vol. I, Section III, chapters 15 and 16 for bacteria and yeasts, Vol. II, Section VI, chapter 82 for viruses, and Vol. II, Section X, chapter 135 for protozoa. As an example of conventional identification methods, mention can be made of the determination of the biological profile, by using the Vitek 2 (bioMerieux) identification cards, for example, or even by using molecular biology techniques with identification criteria based on the study of the presence of certain genes, and on the study of their sequence.
Identification can be performed directly from the sample in which the identification is made, or the microorganisms contained in the sample can be cultured using methods well known to the person skilled in the art with optimal culture media and culturing conditions tailored to the species of microorganisms to be sought, as described by Murray P. R. et al. in Manual of Clinical Microbiology, 2007, 9th edition, Vol. I, Section III, chapter 14, and in particular in Vol. I, Section IV, chapter 21 for bacteria, and Vol. II, Section VI, chapter 81 for viruses, Vol. II, Section VIII, chapter 117 for yeasts, and Vol. II, Section X, chapter 134 for protozoa.
Thus, generally, in the case of an identification using a biochemical method of a bacterium in a specimen, it is first necessary to obtain it in a pure culture, for example after seeding on agar. Molecular biology (FOR) can in certain cases be applied directly to the sample to be analysed.
Instead of cultivating the microorganisms, they can be concentrated by capture directly in the sample by means of active surfaces. Such a method was described by W.-J. Chen et al. [11] who captured different bacterial species with the aid of magnetic beads with an Fe3O4/TiO2-activated surface. Capture by other means is also possible, such as a capture by lectins [23], or by antibodies [24], or by Vancomycin [25]. The capture makes it possible to concentrate the microorganisms and thus to reduce or even eliminate the culture step. This results in a considerable time saving.
The identification may also be performed by mass spectrometry, in accordance with the techniques described previously, preferably by MS, by MS/MS, or even by MS followed by MS/MS spectrometry, which constitutes one embodiment of the invention. In this case too, the sample can be subjected to a culture step beforehand, such as seeding on agar.
The use of an MS identification method is advantageous in that it may be carried out in a few minutes, and in that it requires a mass spectrometer with a single analyser, i.e. a less complex instrument than a tandem mass spectrometer used in MS/MS.
The use of a method of identification by MS followed by MS/MS spectrometry is also advantageous. It makes it possible to check the identity of the ions observed by MS, which increases the specificity of the analysis.
The use of an MRM-type MS/MS identification method has the advantage of being more sensitive and simpler than the conventional MS followed by MS/MS approaches. This method requires neither a high-performance software to process the information between the acquisition of the MS spectrum and of the MS/MS spectrum, nor a change in the setting of the machine parameters for linking up MS then MS/MS spectra.
The method of identification by MS may be employed with an electrospray source on a raw sample, as described by S. Vaidyanathan et al. [26] or by R. Everley et al. [27] after chromatographic separation. Different m/z ranges thus make it possible to identify the microorganisms. S. Vaidyanathan et al. used a window of between 200 and 2000 Th, and R. Everley et al. used a window of between 620 and 2450 Th. The mass spectra may also be deconvoluted to access the mass of the proteins independently of their charge state. R. Everley et al. therefore used masses of between about 5,000 and 50,000 Da. Alternatively, the method of identification by MS can also be employed with the aid of a MALDI-TOF, as described by Claydon et al. [3] and T. Krishnamurthy and P. Ross [4]. The analysis combines acquisition of a mass spectrum and interpretation of expert software. It is extremely simple and can be carried out in a few minutes. This method of identification is currently becoming more widespread in medical analysis laboratories [28].
The identification of bacteria by MS followed by MS/MS via their proteins present in the sample has been applied widely by a number of teams. By way of example, mention can be made of the recent work of Manes N. et al. [29], who studied the peptidome of Salmonella enterica, or the work of R. Nandakumar et al. [30] or of L. Hernychova et al. [31] who have studied the proteome of bacteria after digestion of the proteins with trypsin. The conventional approach consists in i) acquiring an MS spectrum, ii) successively selecting each precursor ion observed on the MS spectrum with an intense signal, iii) successively fragmenting each precursor ion and acquiring its MS/MS spectrum, iv) interrogating protein databases such as SWISS-PROT or NCBI, through software such as Mascot (Matrix Science, London, United Kingdom) or SEQUEST (Thermo Scientific, Waltham, United States of America), to identify the peptide which has a strong probability of matching the MS/MS spectrum observed. This method may lead to the identification of a microorganism if a protein or a peptide characteristic of the species is identified.
One of the advantages of the use of the mass spectrometry lies in that it is particularly useful for quantifying molecules, in the present case the markers of the mechanisms of bacterial resistance to beta-lactams. To this end, the current intensity detected is used, which is proportional to the quantity of target molecule. The current intensity thus measured may serve as a quantitative measurement making it possible to determine the quantity of target molecule present, which is characterised by its expression in International System (SI) mol/m3 or kg/m3 units, or by multiples or sub-multiples of these units, or by the usual derivatives of the SI units, including multiples or sub-multiples thereof. As a non-limiting example, the units such as ng/ml or fmoI/I are units characterising a quantitative measurement.
A calibration is nevertheless necessary in order to be able to correlate the measured area of the peak, which corresponds to the current intensity induced by the detected ions, to the quantity of target molecule to be assayed. For this purpose, the calibrations conventionally used in mass spectrometry may be employed, within the framework of the invention. MRM assays are conventionally calibrated with the aid of external standards or, preferably, with the aid of internal standards such as described by T. Fortin et al. [13]. If the target molecule is a proteotypic peptide which permits the assaying of a protein of interest, the correlation between the quantitative measurement and the quantity of target proteotypic peptide, and subsequently of protein of interest, is obtained by calibrating the measured signal relative to a standard signal for which the quantity to be assayed is known. The calibration may be performed using a calibration curve, for example obtained by successive injections of standard proteotypic peptide at different concentrations (external calibration), or preferably by internal calibration using a heavy peptide as an internal standard, for example in accordance with the AQUA, QconCAT or PSAQ methods detailed below. “Heavy peptide” is understood to mean a peptide corresponding to the proteotypic peptide, but in which one or more atoms of carbon 12 (12C) is (are) replaced by carbon 13 (13C), and/or one or more atoms of nitrogen 14 (14N) is (are) replaced by nitrogen 15 (15N).
The use of heavy peptides as internal standards (AQUA) was also proposed in US patent application 2004/0229283. The principle is to artificially synthesise proteotypic peptides with amino acids containing isotopes which are heavier than the usual natural isotopes. Such amino acids are obtained, for example, by replacing some of the atoms of carbon 12 (12C) with carbon 13 (13C), or by replacing some of the atoms of nitrogen 14 (14N) with nitrogen 15 (15N). The artificial peptide (AQUA) thus synthesised has strictly the same physicochemical properties as the natural peptide (with the exception of a higher mass). It is generally added, at a given concentration, to the sample, upstream of assaying by mass spectroscopy, for example between the treatment entailing the cleaving of the proteins in the sample of interest and the fractionation of the peptides obtained after the treatment step. Thus, the AQUA peptide is co-purified with the natural peptide to be assayed, during fractionation of the peptides. The two peptides are therefore injected simultaneously into the mass spectrometer, for assaying. They then undergo the same ionisation yield in the source. The comparison of the peak areas of the natural and AQUA peptides, whose concentration is known, makes it possible to calculate the concentration of the natural peptide and thus trace back the concentration of the protein to be assayed. A variation of the AQUA technique was proposed by J.-M. Pratt et al. [32] under the name QconCat. This variant is also described in patent application WO 2006/128492.
It consists in concatenating various AQUA peptides and producing the artificial polypeptide in the form of a heavy recombinant protein. The recombinant protein is synthesised with amino acids comprising heavy isotopes. In this way, it is possible to obtain a standard to calibrate the simultaneous assay of several proteins at lower cost. The QconCAT standard is added from the start, upstream of the treatment entailing the cleaving of the proteins and prior to the steps of protein fractionation, denaturation, reduction and blocking of the protein thiol functions, if these are present. The QconCAT standard therefore undergoes the same treatment cycle entailing the cleaving of the proteins as the natural protein, which makes it possible to take account of the yield from the treatment step which entails the cleaving of the proteins. In fact, the treatment, particularly by digestion, of the natural protein may not be complete. In this case, the use of an AQUA standard would lead to underestimating the quantity of natural protein. For full assaying, it may therefore be important to take into account the yields from treatment which entails the cleaving of the proteins. However, V. Brun et al. [33] have shown that the QconQAT standards sometimes do not exactly reproduce the treatment yield, particularly by digestion of the natural protein, undoubtedly due to a three-dimensional conformation different from the QconCAT protein.
V. Brun et al. [33] then proposed the use of a method dubbed PSAQ, and described in patent application WO 2008/145763. In this case, the internal standard is a recombinant protein having the same sequence as the natural protein but synthesised with heavy amino acids. The synthesis is performed ex-vivo with heavy amino acids. This standard has strictly the same physicochemical properties as the natural protein (with the exception of a higher mass). It is added from the start, before the protein fractionation step, when the latter is present. It is therefore co-purified with the native protein, during the protein fractionation step. It exhibits the same treatment yield, particularly by digestion, as the native protein. The heavy peptide obtained after cleaving is also co-purified with the natural peptide, if a peptide fractionation step is performed. The two peptides are therefore injected simultaneously into the mass spectrometer, to be quantitatively assayed. They then undergo the same ionisation yield in the source. Comparison of the peak areas of the natural and the reference peptides in the PSAQ method make it possible to calculate the concentration of the protein to be assayed taking into account all of the steps of the assay method.
All of these techniques, namely AQUA, QconCAT or PSAQ or any other calibration technique, used in the mass spectrometry assays and in particular in MRM or MS assays, may be employed to carry out calibration, within the framework of the invention.
Preferably, the mass spectrometry used in the detection method according to the invention is MS/MS. More preferably, the mass spectrometry is MRM.
The method of the invention makes it possible to detect resistances to cephalosporin, characterised by the detection of at least one peptide as a resistance marker. Said resistance marker peptide preferably belongs to the proteins TEM, CMY, CTX-M, SHV, FOX, ACC, ACT, GARB, DHA, MIR, MOX, PER, VEB, OXA or GES.
In particular, the detection of a mechanism of resistance to cephalosporins induced by the expression of the TEM protein is characterised by the detection of at least one peptide belonging to the TEM protein and its different sequence variants SEQ ID No. 1 to SEQ ID No. 165 and SEQ ID No. 1836 to SEQ ID No. 1843.
said peptides being chosen, preferably, from the peptides of sequence SEQ ID No. 166 to SEQ ID 261 and SEQ ID No. 1923 to SEQ ID 1928 as defined hereafter:
In the clinical interest column, the entries 2b, 2br, 2be and 2ber correspond to the functional subgroups of TEM beta-lactamases which the corresponding peptide makes it possible to detect. Thus, the detection of a 2be peptide indicates the presence of a beta-lactamase with an extended spectrum (ESBL) capable of hydrolysing penicillins, first-generation cephalosporins such as cephaloridine and cefalotin, and at least one antibiotic from the oxyimino-beta-lactam class such as cefotaxime, ceftazidime or monobactams such as aztreonam.
The entry TEM indicates a common peptide between at least two of the subgroups 2b, 2br and 2be or 2ber. The corresponding peptide indicates the presence of a TEM beta-lactamase and the presence of a mechanism of resistance to at least penicillins and first-generation cephalosporins.
The detection of a mechanism of resistance to ESBL (extended-spectrum beta-lactamase) cephalosporins, induced by the TEM protein, is characterised by the detection of at least one resistance-marking 2be peptide, chosen from the sequences SEQ ID No. 168, SEQ ID No. 169, SEQ ID No. 179, SEQ ID No. 181, SEQ ID No. 186, SEQ ID No. 199, SEQ ID No. 200, SEQ ID No. 205, SEQ ID No. 206, SEQ ID No. 208, SEQ ID No. 212, SEQ ID No. 213, SEQ ID No. 214, SEQ ID No. 215, SEQ ID No. 216, SEQ ID No. 217, SEQ ID No. 222, SEQ ID No. 225, SEQ ID No. 226, SEQ ID No. 227, SEQ ID No. 230, SEQ ID No. 234, SEQ ID No. 236, SEQ ID No. 237, SEQ ID No. 238, SEQ ID No. 239, SEQ ID No. 240, SEQ ID No. 249, SEQ ID No. 254, SEQ ID No. 255, SEQ ID No. 256, SEQ ID No. 259, SEQ ID No. 261, SEQ ID No. 1927, SEQ ID No. 1928.
The detection of a mechanism of resistance to cephalosporins induced by the expression of the CMY protein is characterised by the detection of at least one peptide belonging to the CMY protein and to its different sequence variants SEQ ID No. 262 to SEQ ID No. 311 and SEQ ID No. 1844 to SEQ ID No. 1870.
said peptides being chosen, preferably, from the peptides of sequence SEQ ID No. 312 to SEQ ID No. 350, SEQ ID No. 734, SEQ ID No. 735 and SEQ ID No. 1929 to SEQ ID No. 2007, as defined hereafter:
The detection of a mechanism of resistance to cephalosporins induced by the expression of the CTX-M protein is characterised by the detection of at least one peptide belonging to the CTX-M protein and to its different sequence variants SEQ ID No. 351 to SEQ ID No. 445 and SEQ ID No. 1871 to SEQ ID No. 1908.
said peptides being chosen, preferably, from the peptides of sequence SEQ ID No. 446 to SEQ ID 495 and SEQ ID No. 2008 to SEQ ID No. 2092 as defined hereafter:
In the clinical interest column, the entry 2be indicates that all of the CTX-M peptides indicates the presence of a beta-lactamase with an extended spectrum (ESBL) capable of hydrolysing penicillins, first-generation cephalosporins such as cephaloridine and cefalotin, and at least one antibiotic from the oxyimino-beta-lactam class such as cefotaxime, ceftazidime or monobactams such as aztreonam.
The detection of a mechanism of resistance to ESBL (extended-spectrum beta-lactamase) cephalosporins, induced by the CTX-M protein is characterised by the detection of at least one resistance-marking 2be peptide chosen from the sequences SEQ ID No. 446 to SEQ ID No. 478, SEQ ID No. 480 to SEQ ID No. 495 and SEQ ID No. 2008 to SEQ ID No. 2092.
The detection of a mechanism of resistance to cephalosporins induced by the expression of the SHV protein is characterised by the detection of at least one peptide belonging to the SHV protein and to its different sequence variants SEQ ID No. 496 to SEQ ID No. 613 and SEQ ID No. 1909 to SEQ ID No. 1919.
said peptides being chosen, preferably, from the peptides of sequence SEQ ID No. 614 to SEQ ID No. 711 and SEQ ID No. 2093 to SEQ ID No. 2096 as defined hereafter:
In the clinical interest column, the entries 2b, 2br, 2be and 2ber correspond to the functional subgroups of the SHV beta-lactamases which the corresponding peptide makes it possible to detect. Thus, the detection of a 2be peptide will indicate the presence of a beta-lactamase with an extended spectrum (ESBL) capable of hydrolysing penicillins, first-generation cephalosporins such as cephaloridine and cefalotin, and at least one antibiotic from the oxyimino-beta-lactam class such as cefotaxime, ceftazidime or monobactams such as aztreonam.
The entry SHV indicates a common peptide between at least two of the subgroups 2b, 2br and 2be or 2ber. The corresponding peptide indicates the presence of an SHV beta-lactamase and the presence of a mechanism of resistance at least to penicillins and to first-generation cephalosporins.
The detection of a mechanism of resistance to ESBL (extended-spectrum beta-lactamase) cephalosporins, induced by the SHV protein, is characterised by the detection of at least one resistance-marking 2be peptide, chosen from the sequences SEQ ID No. 616, SEQ ID No. 617, SEQ ID No. 627, SEQ ID No. 629, SEQ ID No. 634, SEQ ID No. 647, SEQ ID No. 648, SEQ ID No. 653, SEQ ID No. 654, SEQ ID No. 656, SEQ ID No. 660, SEQ ID No. 661, SEQ ID No. 662, SEQ ID No. 663, SEQ ID No. 664, SEQ ID No. 665, SEQ ID No. 670, SEQ ID No. 673, SEQ ID No, 674, SEQ ID No. 675, SEQ ID No. 678, SEQ ID No. 682, SEQ ID No. 684, SEQ ID No. 685, SEQ ID No. 686, SEQ ID No. 687, SEQ ID No. 688, SEQ ID No. 697, SEQ ID No. 702, SEQ ID No. 703, SEQ ID No. 704, SEQ ID No. 707, SEQ ID No. 708, SEQ ID No. 709, SEQ ID No. 711, SEQ ID No. 2095.
The detection of a mechanism of resistance to cephalosporins induced by the expression of the FOX protein is characterised by the detection of at least one peptide belonging to the FOX protein and to its different sequence variants SEQ ID No. 712 to SEQ ID No. 718 and SEQ ID No. 1920 to SEQ ID No. 1922.
said peptides being chosen, preferably, from the peptides of sequence SEQ ID No. 719 to SEQ ID No. 733 and SEQ ID No. 2097 to SEQ ID No. 2113 as defined hereafter:
AHYFNYGVANR
AMGEQR
ESGQR
FAVPK
GGFELDDK
GIAIVMLANR
IPGMAVAVLK
NYPIEAR
SWSPVYPAGTHR
TGSADLLK
TGSTGGFGAYVAFVPAR
TLTATLGAYAAVK
VSEQTLFEIGSVSK
VSQHAPWLK
VTPGVLAAEAYGIK
The detection of a mechanism of resistance to cephalosporins induced by the expression of the ACC protein is characterised by the detection of at least one peptide belonging to the ACC protein and to its different sequence variants SEQ ID No. 736 to SEQ ID No. 739.
said peptides being chosen, preferably, from the peptides of sequence SEQ ID No. 740 to SEQ ID No. 787 as defined hereafter:
The detection of a mechanism of resistance to cephalosporins induced by the expression of the ACT protein is characterised by the detection of at least one peptide belonging to the ACT protein and to its different sequence variants SEQ ID No. 788 to SEQ ID No. 794.
said peptides being chosen, preferably, from the peptides of sequence SEQ ID No. 795 to SEQ ID No. 841 as defined hereafter:
The detection of a mechanism of resistance to cephalosporins induced by the expression of the CARB protein is characterised by the detection of at least one peptide belonging to the GARB protein and to its different sequence variants SEQ ID No. 842 to SEQ ID No. 852.
said peptides being chosen, preferably, from the peptides of sequence SEQ ID No. 853 to SEQ ID No. 921 as defined hereafter:
The detection of a mechanism of resistance to cephalosporins induced by the expression of the DHA protein is characterised by the detection of at least one peptide belonging to the DHA protein and to its different sequence variants SEQ ID No. 922 to SEQ ID No. 927.
said peptides being chosen, preferably, from the peptides of sequence SEQ ID No. 928 to SEQ ID No. 948 as defined hereafter:
The detection of a mechanism of resistance to cephalosporins induced by the expression of the MIR protein is characterised by the detection of at least one peptide belonging to the MIR protein and to its different sequence variants SEQ ID No. 949 to SEQ ID No. 953.
said peptides being chosen, preferably, from the peptides of sequence SEQ ID No. 954 to SEQ ID No. 981 as defined hereafter:
The detection of a mechanism of resistance to cephalosporins induced by the expression of the MOX protein is characterised by the detection of at least one peptide belonging to the MOX protein and to its different sequence variants SEQ ID No. 982 to SEQ ID No. 988.
said peptides being chosen, preferably, from the peptides of sequence SEQ ID No. 989 to SEQ ID No. 1037 as defined hereafter:
The detection of a mechanism of resistance to cephalosporins induced by the expression of the PER protein is characterised by the detection of at least one peptide belonging to the PER protein and to its different sequence variants SEQ ID No. 1038 to SEQ ID No. 1044.
said peptides being chosen, preferably, from the peptides of sequence SEQ ID No. 1045 to SEQ ID No. 1077 as defined hereafter:
The detection of a mechanism of resistance to cephalosporins induced by the expression of the PER protein indicates an ESBL resistance. It is preferably detected with the aid of SEQ ID No. 1045 to SEQ ID No. 1065 and SEQ ID No. 1069 to SEQ ID No. 1077.
The detection of a mechanism of resistance to cephalosporins induced by the expression of the VEB protein is characterised by the detection of at least one peptide belonging to the VEB protein and to its different sequence variants SEQ ID No. 1078 to SEQ ID No. 1084.
said peptides being chosen, preferably, from the peptides of sequence SEQ ID No. 1085 to SEQ ID No. 1104 as defined hereafter:
The detection of a mechanism of resistance to cephalosporins induced by the expression of the VEB protein indicates an ESBL resistance.
The detection of a mechanism of resistance to cephalosporins or to carbapenems induced by the expression of the OXA protein is characterised by the detection of at least one peptide belonging to the OXA protein and to its different sequence variants SEQ ID No. 1105 to SEQ ID No. 1266:
said peptides being chosen, preferably, from the peptides of sequence SEQ ID No. 1267 to SEQ ID No. 1835 and SEQ ID No. 2160 to SEQ ID No. 2171 as defined hereafter:
In the clinical interest column, the entries 2d, 2de, 2df correspond to the functional subgroups of OXA beta-lactamases which the corresponding peptide makes it possible to detect. Therefore, the detection of a 2df peptide will indicate the presence of a carbapenemase beta-lactamase capable of hydrolysing carbapenems.
The entry 2de will indicate the presence of a beta-lactamase with an extended spectrum (ESBL) capable of hydrolysing penicillins, first-generation cephalosporins such as cephaloridine and cefalotin, and at least one antibiotic from the oxyimino-beta-lactam class such as cefotaxime, ceftazidime or monobactams such as aztreonam.
The entry OXA indicates a common peptide between at least two of the subgroups 2d, 2de and 2df. The corresponding peptide indicates the presence of an OXA beta-lactamase and the presence of a mechanism of resistance at least to penicillins and to first-generation cephalosporins.
The detection of a mechanism of resistance to ESBL (extended-spectrum beta-lactamase) cephalosporins, induced by the OXA protein, is characterised by the detection of at least one resistance-marking 2de peptide chosen from the sequences SEQ ID No, 1277, 1297, 1323, 1344, 1368, 1369, 1373, 1375, 1392, 1421, 1423, 1427, 1429, 1439, 1444, 1446, 1450, 1451, 1457, 1465, 1468, 1490, 1491, 1495, 1498, 1534, 1543, 1545, 1575, 1589, 1597, 1598, 1600, 1602, 1657, 1713, 1721, 1724, 1740, 1742, 1787, 1824, 1268, 1269, 1270, 1271, 1272, 1278, 1279, 1280, 1281, 1283, 1285, 1288, 1290, 1295, 1299, 1300, 1301, 1302, 1303, 1304, 1305, 1306, 1307, 1308, 1309, 1310, 1314, 1315, 1316, 1317, 1318, 1319, 1320, 1332, 1337, 1339, 1340, 1341, 1342, 1350, 1354, 1355, 1356, 1357, 1358, 1359, 1360, 1365, 1366, 1367, 1386, 1389, 1390, 1391, 1393, 1394, 1402, 1404, 1405, 1407, 1408, 1413, 1414, 1419, 1420, 1425, 1432, 1433, 1440, 1447, 1448, 1456, 1471, 1472, 1477, 1478, 1480, 1485, 1487, 1488, 1499, 1504, 1506, 1508, 1509, 1510, 1513, 1514, 1515, 1516, 1521, 1522, 1525, 1526, 1530, 1533, 1539, 1540, 1548, 1550, 1551, 1552, 1553, 1554, 1555, 1556, 1559, 1567, 1569, 1570, 1571, 1572, 1574, 1577, 1582, 1590, 1592, 1595, 1596, 1605, 1609, 1610, 1611, 1612, 1613, 1614, 1615, 1616, 1617, 1618, 1620, 1626, 1627, 1628, 1632, 1633, 1634, 1635, 1637, 1638, 1639, 1640, 1652, 1653, 1656, 1660, 1661, 1662, 1664, 1665, 1666, 1669, 1670, 1671, 1672, 1673, 1677, 1678, 1680, 1681, 1685, 1687, 1695, 1696, 1697, 1703, 1704, 1706, 1707, 1708, 1709, 1712, 1714, 1715, 1717, 1720, 1722, 1725, 1727, 1729, 1730, 1732, 1733, 1737, 1738, 1743, 1746, 1748, 1751, 1752, 1753, 1754, 1755, 1758, 1759, 1760, 1761, 1762, 1763, 1764, 1765, 1769, 1771, 1773, 1775, 1776, 1777, 1781, 1785, 1788, 1790, 1791, 1792, 1793, 1794, 1795, 1796, 1797, 1798, 1800, 1805, 1806, 1809, 1810, 1815, 1816, 1818, 1828, 2160, 2161, 2162, 2163, 2164, 2165, 2166, 2167, 2168, 2169, 2170, 2171.
The detection of a mechanism of resistance to carbapenems or to cephalosporins, induced by the expression of the GES protein, is characterised by the detection of at least one peptide belonging to the GES protein and to its different sequence variants SEQ ID No. 2114 to SEQ ID No. 2130.
said peptides being chosen, preferably, from the peptides of sequence SEQ ID No, 2131 to SEQ ID No. 2159 as defined hereafter:
In the clinical interest column, the ESBL and carba entries correspond to the GES beta-lactamase activities which the corresponding peptide makes it possible to detect. Therefore, the detection of a carba peptide will indicate the presence of a carbapenemase beta-lactamase capable of hydrolysing carbapenems, penicillins, first-generation cephalosporins such as cephaloridine and cefalotin, and at least one antibiotic from the oxyimino-beta-lactam class such as cefotaxime, ceftazidime or the monobactames such as aztreonam.
If no peptide referred to as carba is detected, the detection of a peptide referred to as ESBL will indicate the presence of a beta-lactamase with an extended spectrum (ESBL) capable of hydrolysing penicillins, first-generation cephalosporins such as cephaloridine and cefalotin, and at least one antibiotic from the oxyimino-beta-lactam class such as cefotaxime, ceftazidime or monobactams such as aztreonam.
The detection of a mechanism of resistance to cephalosporinases with an extended spectrum (ESBL) induced by the GES protein is thus characterised by the detection of at least one resistance-marking ESBL peptide chosen from the sequences SEQ ID No. 2131 to SEQ ID No. 2159.
Certain peptide sequences can be common to several resistance mechanisms. Therefore, the following sequences are identical:
SEQ ID No. 834 and SEQ ID No. 978
SEQ ID No. 833 and SEQ ID No. 977
SEQ ID No. 832 and SEQ ID No. 976
SEQ ID No. 831 and SEQ ID No. 975
SEQ ID No. 826 and SEQ ID No. 974
SEQ ID No. 825 and SEQ ID No. 973
SEQ ID No. 824 and SEQ ID No. 972
SEQ ID No. 823 and SEQ ID No. 971
SEQ ID No. 822 and SEQ ID No. 970
SEQ ID No. 817 and SEQ ID No. 969
SEQ ID No. 813 and SEQ ID No. 967
SEQ ID No. 802 and SEQ ID No. 964
SEQ ID No. 798 and SEQ ID No. 963
SEQ ID No. 666 and SEQ ID No. 895
SEQ ID No, 350 and SEQ ID No. 1035
SEQ ID No. 349 and SEQ ID No. 1031
SEQ ID No. 347 and SEQ ID No. 1030
SEQ ID No, 346 and SEQ ID No. 1029
SEQ ID No. 345 and SEQ ID No. 1028
SEQ ID No. 343 and SEQ ID No. 1037 and SEQ ID No. 725
SEQ ID No. 342 and SEQ ID No. 1026
SEQ ID No. 341 and SEQ ID No. 1025
SEQ ID No. 340 and SEQ ID No. 1024
SEQ ID No. 339 and SEQ ID No. 1023
SEQ ID No. 338 and SEQ ID No. 1020
SEQ ID No. 337 and SEQ ID No. 1036 and SEQ ID No. 719
SEQ ID No. 337 and SEQ ID No. 1036
SEQ ID No. 336 and SEQ ID No. 841 and SEQ ID No. 981
SEQ ID No. 323 and SEQ ID No. 840 and SEQ ID No. 980
SEQ ID No. 315 and SEQ ID No. 839 and SEQ ID No. 948 and SEQ ID No. 979
SEQ ID No. 240 and SEQ ID No. 679
SEQ ID No. 184 and SEQ ID No. 626
SEQ ID No. 182 and SEQ ID No. 625
SEQ ID No. 1034 and SEQ ID No. 1999
SEQ ID No. 1032 and SEQ ID No. 1988
SEQ ID No. 1027 and SEQ ID No. 1970
SEQ ID No. 1022 and SEQ ID No. 1952
SEQ ID No. 1021 and SEQ ID No. 1937
SEQ ID No. 1019 and SEQ ID No. 1933
SEQ ID No. 809 and SEQ ID No. 966
SEQ ID No. 1033 and SEQ ID No. 1989
In all cases, the sequences above indicate the expression of a mechanism of resistance to cephalosporins.
The method of the invention and its advantages will become apparent from the rest of the present description which presents several non-limiting examples of implementation of said method.
1. Culturing of the Sample on a Culture Medium
The optimum culture media and the optimum culture conditions are different according to the species of microorganism. By default, the sample is seeded on different media:
2. Identification of the Microorganisms
The identification is performed as follows:
The following protocol is performed in 16 steps (steps 5 to 12 are optional and could be omitted if the enriched sample is subsequently treated according to examples 4 and onwards):
The identification is performed as follows:
The identification is performed as follows:
The following protocol is conventionally performed in 17 steps:
Samples Sam10 to Sam61 are identified according to one of the methods described in examples 1, 3 or 4. The identification of the species is set out in TABLE 1.
P. mirabilis
E. coli
E. aerogenes
E. coli
E. coli
E. coli
S. marcescens
E. coli
E. coli
P. mirabilis
C. freundii
E. coli
P. mirabilis
E. coli
K. pneumoniae
E. coli
K. oxytoca
P. mirabilis
E. coli
P. mirabilis
P. rettgeri
P. stuartii
S. Derby
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
E. coli
K. pneumoniae
E. coli
K. pneumoniae
P. mirabilis
P. mirabilis
P. mirabilis
P. mirabilis
P. mirabilis
K. pneumoniae
P. mirabilis
E. coli
E. coli
P. mirabilis
E. aerogenes
E. aerogenes
E. coli
E. coli
Samples Sam10 to Sam61 correspond to a species able to comprise a TEM resistance mechanism (Enterobacteriaceae . . . ). The following method is then performed to search for such a mechanism.
Each sample is treated according to example 5, then a volume of 50 μl of digested proteins is injected and analysed according to the following conditions:
The transitions mentioned in TABLE 3 are detected by using the parameters set out in TABLE 4. The precursor peptide retention time and the transitions, i.e. the (m/z) 1 ratio in Q1 and (m/z)2 ratio in Q3, as well as the collision energy used to fragment the precursor ion, are indicated in TABLE 4. The threshold above which the transition is considered to be detected is also indicated in TABLE 4.
The other machine parameters used are as follows:
The areas obtained for each of the transitions and for each of the microorganisms studied were measured. When the areas of the 3 transitions of the same peptide are greater than or equal to the positivity threshold described in TABLE 4, the detection of the peptide is considered to be positive and is labelled “1” in TABLE 5. When at least one transition comprises an area less than the positivity threshold described in TABLE 4, the corresponding peptide is considered non-detected and is labelled 0 in TABLE 5.
Sample Sam10 does not present any peptide which is characteristic of TEMs. The bacteria present in sample Sam10 may be sensitive to cephalosporins and to penicillins.
Samples Sam11 to Sam61 comprise at least one peptide which is characteristic of TEMs. The bacteria present in samples Sam11 to Sam61 therefore express a beta-lactamase which confers on them a resistance to penicillins.
No peptide specific to phenotype 2b is observed, and no sample tested is identified as being resistant solely to penicillins.
Samples Sam35, Sam49 and Sam50 comprise at least one peptide specific to phenotype 2br. Samples Sam35, Sam49 and Sam50 are therefore resistant to penicillins associated with an inhibitor of the clavulanic acid and tazobactam type.
No peptide specific to phenotype 2ber is observed, and no sample tested is identified as belonging only to this phenotype.
Samples Sam12 to Sam21, Sam23 to Sam27, Sam29, Sam32, Sam33, Sam44, Sam46, Sam52, Sam55 and Sam57 to Sam61 comprise at least one peptide specific to the phenotype 2be or to the phenotype 2ber. Samples Sam12 to Sam21, Sam23 to Sam27, Sam29, Sam32, Sam33, Sam44, Sam46, Sam52, Sam55, and Sam57 to Sam61 are therefore resistant to penicillins, to cephalosporins and to monobactams.
The samples corresponding to a species able to comprise a CMY resistance mechanism can be detected by employing the following method.
Each sample is treated according to example 5, then analysed according to example 6 by detecting the peptides from TABLE 6 instead of the peptides from TABLE 3.
The areas obtained for each of the transitions and for each of the microorganisms studied were measured. When the areas of the 3 transitions of the same peptide are greater than or equal to 2500, the detection of the peptide is considered to be positive and is labelled “1”. When at least one transition comprises an area less than 2500, the corresponding peptide is considered non-detected and is labelled “0”.
The samples corresponding to a species able to comprise a CTX-M resistance mechanism can be detected by employing the following method.
Each sample is treated according to example 5, then analysed according to example 6 by detecting the peptides from TABLE 7 instead of the peptides from TABLE 3.
The areas obtained for each of the transitions and for each of the microorganisms studied were measured. When the areas of the 3 transitions of the same peptide are greater than or equal to 2500, the detection of the peptide is considered to be positive and is labelled “1”. When at least one transition comprises an area less than 2500, the corresponding peptide is considered non-detected and is labelled “0”.
The samples corresponding to a species able to comprise an SHV resistance mechanism can be detected by employing the following method.
Each sample is treated according to example 5, then analysed according to example 6 by detecting the peptides from TABLE 8 instead of the peptides from TABLE 3.
The areas obtained for each of the transitions and for each of the microorganisms studied were measured. When the areas of the 3 transitions of the same peptide are greater than or equal to 2500, the detection of the peptide is considered to be positive and is labelled “1”. When at least one transition comprises an area less than 2500, the corresponding peptide is considered non-detected and is labelled “0”.
The samples corresponding to a species able to comprise a FOX resistance mechanism can be detected by employing the following method.
Each sample is treated according to example 5, then analysed according to example 6 by detecting the peptides from TABLE 9 instead of the peptides from TABLE 3.
The areas obtained for each of the transitions and for each of the microorganisms studied were measured. When the areas of the three transitions of the same peptide are greater than or equal to 2500, the detection of the peptide is considered to be positive and is labelled “1”. When at least one transition comprises an area less than 2500, the corresponding peptide is considered non-detected and is labelled “0”.
The samples corresponding to a species able to comprise a TEM or CTX-M resistance mechanism can be detected by employing the following method.
Each sample is treated according to example 5, then analysed according to example 6 by detecting the peptides from TABLE 10 instead of the peptides from TABLE 3.
In the clinical interest column, the entries TEM, 2b, 2br, 2be and 2ber correspond to the same meanings as in TABLE 4.
The transitions mentioned in TABLE 10 are detected by using the parameters set out in TABLE 11.
When the areas of at least two transitions of the same peptide are greater than or equal to the positivity threshold described in TABLE 11, the detection of the peptide is considered to be positive. When more than two transitions of the same peptide comprise an area less than the positivity threshold described in TABLE 11, the corresponding peptide is considered non-detected.
A sample contains bacteria which express the TEM protein, when at least one peptide corresponding to the TEM resistance mechanism is detected. These bacteria are resistant to penicillins.
A sample contains bacteria which express the TEM protein, phenotype 2b, when at least one peptide corresponding to the TEM resistance mechanism clinical interest 2b is detected. These bacteria are only resistant to penicillins.
A sample contains bacteria which express the TEM protein, phenotype 2br, when at least one peptide corresponding to the TEM resistance mechanism clinical interest 2br is detected. These bacteria are resistant to penicillins associated with an inhibitor of the clavulanic acid and tazobactam type.
A sample contains bacteria which express the CTX-M protein or the TEM protein, phenotype 2be, when at least one peptide corresponding to the CTX-M or TEM resistance mechanism clinical interest 2be is detected. These bacteria are resistant to penicillins, to cephalosporins and to monobactams.
A sample contains bacteria which express the TEM protein, phenotype 2ber, when at least one peptide corresponding to the TEM resistance mechanism clinical interest 2ber, is detected. These bacteria are resistant to penicillins, to cephalosporins and to monobactams, and are insensitive to inhibition by clavulanic acid, sulfobactam or tazobactam.
Samples Sam74 to Sam78 are identified according to one of the methods described in examples 1, 3 or 4. The identification of the species is set out in TABLE 12.
A. baumannii
A. baumannii
P. aeruginosa
P. aeruginosa
P. aeruginosa
Samples Sam74 to Sam78 correspond to a species able to comprise a PER resistance mechanism. Each sample is treated according to example 5, then analysed according to example 6 by detecting the peptides from TABLE 13 instead of the peptides from TABLE 3.
The other machine parameters used are as follows:
The areas obtained for each of the transitions and for each of the microorganisms studied were measured. When the areas of the transitions are greater than or equal to the positivity threshold described in TABLE 13, the detection of the transition is considered to be positive and is labelled “1” in TABLE 14. When a transition has an area less than the positivity threshold described in TABLE 13, the transition is considered non-detected and is labelled “0” in TABLE 14.
For a given peptide, when at least 3 transitions are labelled “1”, the peptide is considered as being detected.
Samples Sam74 to Sam78 comprise at least one peptide which is characteristic of PERs. The bacteria present in samples Sam74 to Sam78 therefore express a beta-lactamase which confers on them a resistance to penicillins, cephalosporins and monobactams.
Samples Sam79 to Sam82 are identified according to one of the methods described in examples 1, 3 or 4. The identification of the species is set out in TABLE 15.
A. baumannii
A. baumannii
A. baumannii
E. coli
Samples Sam79 to Sam82 correspond to a species able to comprise a VEB resistance mechanism. Each sample is treated according to example 5, then analysed according to example 6 by detecting the peptides from TABLE 16 instead of the peptides from TABLE 3.
The other machine parameters used are as follows:
The areas obtained for each of the transitions and for each of the microorganisms studied were measured. When the areas of the transitions are greater than or equal to the positivity threshold described in TABLE 16, the detection of the transition is considered to be positive and is labelled “1” in TABLE 17. When a transition has an area less than the positivity threshold described in TABLE 16, the transition is considered non-detected and is labelled “0” in TABLE 17.
For a given peptide, when at least 3 transitions are labelled “1”, the peptide is considered as being detected.
Samples Sam79 to Sam82 comprise at least one peptide which is characteristic of VEBs. The bacteria present in samples Sam79 to Sam82 therefore express a beta-lactamase which confers on them a resistance to penicillins, cephalosporins and monobactams.
Sample Sam83 is identified according to one of the methods described in examples 1, 3 or 4. The identification of the species is set out in TABLE 18.
E. coli
Sample Sam83 corresponds to a species able to comprise a MOX resistance mechanism. Each sample is treated according to example 5, then analysed according to example 6 by detecting the peptides from TABLE 19 instead of the peptides from TABLE 3.
The other machine parameters used are as follows:
The areas obtained for each of the transitions and for each of the microorganisms studied were measured. When the areas of the transitions are greater than or equal to the positivity threshold described in TABLE 19, the detection of the transition is considered to be positive and is labelled “1” in TABLE 20. When a transition has an area less than the positivity threshold described in TABLE 19, the transition is considered non-detected and is labelled “0” in TABLE 20.
For a given peptide, when at least 3 transitions are labelled “1”, the peptide is considered as being detected.
Sample Sam83 comprises at least one peptide which is characteristic of MOXs. The bacteria present in sample Sam83 therefore express a beta-lactamase which confers on them a resistance to penicillins and to cephalosporins, with the exception of fourth-generation cephalosporins.
The samples corresponding to a species able to comprise an ACC resistance mechanism can be detected by employing the following method.
Each sample is treated according to example 5, then analysed according to example 6 by detecting the peptides from TABLE 21 instead of the peptides from TABLE 3.
The areas obtained for each of the transitions and for each of the microorganisms studied were measured. When the areas of the three transitions of the same peptide are greater than or equal to 2500, the detection of the peptide is considered to be positive and is labelled “1”. When at least one transition comprises an area less than 2500, the corresponding peptide is considered non-detected and is labelled “0”.
The samples corresponding to a species able to comprise an ACT resistance mechanism can be detected by employing the following method.
Each sample is treated according to example 5, then analysed according to example 6 by detecting the peptides from TABLE 22 instead of the peptides from TABLE 3.
The areas obtained for each of the transitions and for each of the microorganisms studied were measured. When the areas of the three transitions of the same peptide are greater than or equal to 2500, the detection of the peptide is considered to be positive and is labelled “1”. When at least one transition comprises an area less than 2500, the corresponding peptide is considered non-detected and is labelled “0”.
The samples corresponding to a species able to comprise a CMY resistance mechanism can be detected by employing the following method.
Each sample is treated according to example 5, then analysed according to example 6 by detecting the peptides from TABLE 23 instead of the peptides from TABLE 3.
The areas obtained for each of the transitions and for each of the microorganisms studied were measured. When the areas of the three transitions of the same peptide are greater than or equal to 2500, the detection of the peptide is considered to be positive and is labelled “1”. When at least one transition comprises an area less than 2500, the corresponding peptide is considered non-detected and is labelled “0”.
The samples corresponding to a species able to comprise a CTX-M resistance mechanism can be detected by employing the following method.
Each sample is treated according to example 5, then analysed according to example 6 by detecting the peptides from TABLE 24 instead of the peptides from TABLE 3.
The areas obtained for each of the transitions and for each of the microorganisms studied were measured. When the areas of the three transitions of the same peptide are greater than or equal to 2500, the detection of the peptide is considered to be positive and is labelled “1”. When at least one transition comprises an area less than 2500, the corresponding peptide is considered non-detected and is labelled “0”.
The samples corresponding to a species able to comprise a DHA resistance mechanism can be detected by employing the following method.
Each sample is treated according to example 5, then analysed according to example 6 by detecting the peptides from TABLE 25 instead of the peptides from TABLE 3.
The areas obtained for each of the transitions and for each of the microorganisms studied were measured. When the areas of the three transitions of the same peptide are greater than or equal to 2500, the detection of the peptide is considered to be positive and is labelled “1”. When at least one transition comprises an area less than 2500, the corresponding peptide is considered non-detected and is labelled “0”.
The samples corresponding to a species able to comprise a FOX resistance mechanism can be detected by employing the following method.
Each sample is treated according to example 5, then analysed according to example 6 by detecting the peptides from TABLE 26 instead of the peptides from TABLE 3.
The areas obtained for each of the transitions and for each of the microorganisms studied were measured. When the areas of the three transitions of the same peptide are greater than or equal to 2500, the detection of the peptide is considered to be positive and is labelled “1”. When at least one transition comprises an area less than 2500, the corresponding peptide is considered non-detected and is labelled “0”.
The samples corresponding to a species able to comprise an MIR resistance mechanism can be detected by employing the following method.
Each sample is treated according to example 5, then analysed according to example 6 by detecting the peptides from TABLE 27 instead of the peptides from TABLE 3.
The areas obtained for each of the transitions and for each of the microorganisms studied were measured. When the areas of the three transitions of the same peptide are greater than or equal to 2500, the detection of the peptide is considered to be positive and is labelled “1”. When at least one transition comprises an area less than 2500, the corresponding peptide is considered non-detected and is labelled “0”.
The samples corresponding to a species able to comprise an SHV resistance mechanism can be detected by employing the following method.
Each sample is treated according to example 5, then analysed according to example 6 by detecting the peptides from TABLE 28 instead of the peptides from TABLE 3.
The areas obtained for each of the transitions and for each of the microorganisms studied were measured. When the areas of the three transitions of the same peptide are greater than or equal to 2500, the detection of the peptide is considered to be positive and is labelled “1”. When at least one transition comprises an area less than 2500, the corresponding peptide is considered non-detected and is labelled “0”.
The samples corresponding to a species able to comprise a CARB resistance mechanism can be detected by employing the following method.
Each sample is treated according to example 5, then analysed according to example 6 by detecting the peptides from TABLE 29 instead of the peptides from TABLE 3.
The areas obtained for each of the transitions and for each of the microorganisms studied were measured. When the areas of the three transitions of the same peptide are greater than or equal to 2500, the detection of the peptide is considered to be positive and is labelled “1”. When at least one transition comprises an area less than 2500, the corresponding peptide is considered non-detected and is labelled “0”.
The samples corresponding to a species able to comprise an OXA resistance mechanism can be detected by employing the following method.
Each sample is treated according to example 5, then analysed according to example 6 by detecting the peptides from TABLE 30 instead of the peptides from TABLE 3.
The areas obtained for each of the transitions and for each of the microorganisms studied were measured. When the areas of the three transitions of the same peptide are greater than or equal to 2500, the detection of the peptide is considered to be positive and is labelled “1”. When at least one transition comprises an area less than 2500, the corresponding peptide is considered non-detected and is labelled “0”.
Samples Sam96 to Sam101 are identified according to one of the methods described in examples 1, 3 or 4. The identification of the species is set out in TABLE 31.
K. oxytoca
S. livingstone
Salmonella spp
S. enterica ssp enterica
K. pneumoniae
H. alvei
Samples Sam96 to Sam101 correspond to a species able to comprise an ACC resistance mechanism. Each sample is treated according to example 5, then analysed according to example 6 by detecting the peptides from TABLE 32 instead of the peptides from TABLE 3.
The other machine parameters used are as follows:
The areas obtained for each of the transitions and for each of the microorganisms studied were measured. When the areas of the transitions are greater than or equal to the positivity threshold described in TABLE 32, the detection of the transition is considered to be positive and is labelled “1” in TABLE 33. When a transition has an area less than the positivity threshold described in TABLE 32, the transition is considered non-detected and is labelled “0” in TABLE 33.
For a given peptide, when at least 3 transitions are labelled “1”, the peptide is considered as being detected.
Samples Sam96 to Sam101 comprise at least one peptide which is characteristic of the ACC proteins. The bacteria present in samples Sam96 to Sam101 therefore express a beta-lactamase which confers on them a resistance to penicillins and to cephalosporins, with the exception of fourth-generation cephalosporins.
Samples Sam102 to Sam108 are identified according to one of the methods described in examples 1, 3 or 4. The identification of the species is set out in TABLE 34.
P. mirabilis
S. senftenberg
P. mirabilis
K. oxytoca
E. coli
S. enterica ssp enterica
P. mirabilis
Samples Sam102 to Sam108 correspond to a species able to comprise a CMY resistance mechanism. Each sample is treated according to example 5, then analysed according to example 6 by detecting the peptides from TABLE 35 instead of the peptides from TABLE 3.
The other machine parameters used are as follows:
The areas obtained for each of the transitions and for each of the microorganisms studied were measured. When the areas of the transitions are greater than or equal to the positivity threshold described in TABLE 35, the detection of the transition is considered to be positive and is labelled “1” in TABLE 36. When a transition has an area less than the positivity threshold described in TABLE 35, the transition is considered non-detected and is labelled “0” in TABLE 36.
For a given peptide, when at least 3 transitions are labelled “1”, the peptide is considered as being detected.
Samples Sam102 to Sam108 comprise at least one peptide which is characteristic of the CMY proteins. The bacteria present in samples Sam102 to Sam108 therefore express a beta-lactamase which confers on them a resistance to penicillins and to cephalosporins, with the exception of fourth-generation cephalosporins.
Samples Sam109 to Sam118 are identified according to one of the methods described in examples 1, 3 or 4. The identification of the species is set out in TABLE 37.
E. aerogenes
E. coli
E. coli
E. coli
E. coli
K. pneumoniae
K. pneumoniae
P. mirabilis
Salmonella spp
Salmonella spp
Samples Sam109 to Sam118 correspond to a species able to comprise a CTX-M resistance mechanism. Each sample is treated according to example 5, then analysed according to example 6 by detecting the peptides from TABLE 38 instead of the peptides from TABLE 3.
The other machine parameters used are as follows:
The areas obtained for each of the transitions and for each of the microorganisms studied were measured. When the areas of the transitions are greater than or equal to the positivity threshold described in TABLE 38, the detection of the transition is considered to be positive and is labelled “1” in TABLE 39. When a transition has an area less than the positivity threshold described in TABLE 38, the transition is considered non-detected and is labelled “0” in TABLE 39.
For a given peptide, when at least 3 transitions are labelled “1”, the peptide is considered as being detected.
Samples Sam109 to Sam118 comprise at least one peptide which is characteristic of the CTX-M proteins. The bacteria present in samples Sam109 to Sam118 therefore express a beta-lactamase which confers on them a resistance to penicillins, to cephalosporins and to monobactams.
Samples Sam119 to Sam124 are identified according to one of the methods described in examples 1, 3 or 4. The identification of the species is set out in TABLE 40.
E. coli
K. oxytoca
K. pneumoniae
K. pneumoniae
K. pneumoniae
K. pneumoniae
Samples Sam119 to Sam124 correspond to a species able to comprise a DHA resistance mechanism. Each sample is treated according to example 5, then analysed according to example 6 by detecting the peptides from TABLE 41 instead of the peptides from TABLE 3.
The other machine parameters used are as follows:
The areas obtained for each of the transitions and for each of the microorganisms studied were measured. When the areas of the transitions are greater than or equal to the positivity threshold described in TABLE 41, the detection of the transition is considered to be positive and is labelled “1” in TABLE 42. When a transition has an area less than the positivity threshold described in TABLE 41, the transition is considered non-detected and is labelled “0” in TABLE 42.
For a given peptide, when at least 3 transitions are labelled “1”, the peptide is considered as being detected.
Samples 119 to Sam124 comprise at least one peptide which is characteristic of the DHA proteins. The bacteria present in samples Sam119 to Sam124 therefore express a beta-lactamase which confers on them a resistance to penicillins and to cephalosporins, with the exception of fourth-generation cephalosporins.
Samples Sam125 to Sam130 are identified according to one of the methods described in examples 1, 3 or 4. The identification of the species is set out in TABLE 43.
E. coli
E. coli
K. oxytoca
K. oxytoca
K. pneumoniae
K. pneumoniae
Samples Sam125 to Sam130 correspond to a species able to comprise a FOX resistance mechanism. Each sample is treated according to example 5, then analysed according to example 6 by detecting the peptides from TABLE 44 instead of the peptides from TABLE 3.
The other machine parameters used are as follows:
The areas obtained for each of the transitions and for each of the microorganisms studied were measured. When the areas of the transitions are greater than or equal to the positivity threshold described in TABLE 44, the detection of the transition is considered to be positive and is labelled “1” in TABLE 45, When a transition has an area less than the positivity threshold described in TABLE 44, the transition is considered non-detected and is labelled “0” in TABLE 45.
For a given peptide, when at least 2 transitions are labelled “1”, the peptide is considered as being detected.
Samples Sam125 to Sam130 comprise at least one peptide which is characteristic of the FOX proteins. The bacteria present in samples Sam125 to Sam130 therefore express a beta-lactamase which confers on them a resistance to penicillins and to cephalosporins, with the exception of fourth-generation cephalosporins.
Samples Sam131 to Sam144 are identified according to one of the methods described in examples 1, 3 or 4. The identification of the species is set out in TABLE 46.
E. aerogenes
E. coli
E. coli
E. coli
E. coli
K. pneumoniae
K. pneumoniae
K. pneumoniae
K. pneumoniae
K. pneumoniae
K. pneumoniae
K. pneumoniae
K. pneumoniae
K. pneumoniae
Samples Sam131 to Sam144 correspond to a species able to comprise an SHV resistance mechanism. Each sample is treated according to example 5, then analysed according to example 6 by detecting the peptides from TABLE 47 instead of the peptides from TABLE 3.
The other machine parameters used are as follows:
The areas obtained for each of the transitions and for each of the microorganisms studied were measured. When the areas of the transitions are greater than or equal to the positivity threshold described in TABLE 47, the detection of the transition is considered to be positive and is labelled “1” in TABLE 48. When a transition has an area less than the positivity threshold described in TABLE 47, the transition is considered non-detected and is labelled “0” in TABLE 48.
For a given peptide, when at least 3 transitions are labelled “1”, the peptide is considered as being detected.
Samples Sam131 to Sam144 comprise at least one peptide which is characteristic of the SHV proteins. The bacteria present in samples Sam131 to Sam144 therefore express a beta-lactamase which confers on them a resistance to penicillins.
Sample Sam142 comprises a peptide specific to phenotype 2be. Therefore sample Sam142 is resistant to penicillins, to cephalosporins and to monobactams.
No peptide specific to phenotypes 2b and 2br is observed, no sample tested is identified as belonging only to these phenotypes.
The detection methods described in examples 6 to 30 are particularly advantageous because they make it possible to assay a large number of peptides and at the same time to detect the presence of one or more resistance mechanisms induced by one or more beta-lactamases.
Furthermore, the detection is performed in a short time, less than one hour. In fact, only the part of the gradient between 3 and 34 minutes is useful to the analysis. Furthermore, the retention times of the assayed peptides are all below 34 minutes.
In addition, the detection methods described in examples 6 to 30 are more advantageous than the molecular biology methods because they detect the product of the expression of the genes, and not the genes themselves. The detection of a resistance may not have any clinical meaning if this gene is not expressed, or it if is expressed too weakly to lead to an effective resistance. The detection of a peptide characterising a protein characteristic of a resistance mechanism does not have this disadvantage.
Surprisingly, the above examples show that it is possible to attain by mass spectrometry the sensitivity necessary for the specific detection of the existence of a mechanism of resistance to at least one antimicrobial of a microorganism contained in a sample, without employing an amplification method as is usually the case when molecular biology methods are used.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP2012/057322 | 4/20/2012 | WO | 00 | 12/23/2013 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2012/143534 | 10/26/2012 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 20040229283 | Gygi et al. | Nov 2004 | A1 |
| 20070006950 | Okada et al. | Jan 2007 | A1 |
| 20070269895 | Aebersold | Nov 2007 | A1 |
| 20110245105 | Citri | Oct 2011 | A1 |
| 20120245128 | Haag | Sep 2012 | A1 |
| 20120264156 | Beaulieu et al. | Oct 2012 | A1 |
| Number | Date | Country |
|---|---|---|
| WO 2005098071 | Oct 2005 | WO |
| WO 2006128492 | Dec 2006 | WO |
| WO 2008066629 | Jun 2008 | WO |
| WO 2008145763 | Dec 2008 | WO |
| WO 2011045544 | Apr 2011 | WO |
| Entry |
|---|
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| Number | Date | Country | |
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| 61477907 | Apr 2011 | US |
