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    • 专利摘要:
    The present invention relates to an electrochemical method for the in vitro determination of the presence of beta-lactamase-producing bacteria in a sample capable of containing said bacteria, said method comprising the application of an electrochemical analysis means for determining the presence of the bacteria producing the beta-lactamases.
    公开号:FR3034523A1
    申请号:FR1552928
    申请日:2015-04-03
    公开日:2016-10-07
    发明作者:Murielle Dequaire-Rochelet；Alain Hartmann；Catherine Neuwirth；Benoit Chantemesse
    申请人:Centre Hospitalier Regional Univ De Dijon；Inst Nat de la Rech Agronomique - Inra；Institut National de la Recherche Agronomique INRA；Universite de Bourgogne；
    IPC主号:
    专利说明:

    [0001] 1 NOVEL METHOD FOR DETECTING THE PRESENCE OF BETA-LACTAMASES-PRODUCING BACTERIA The present invention relates to a method for detecting, in a sample or a culture medium, the presence of beta-lactam-resistant bacteria via their ability to produce beta-lactamases. Beta-lactams, which include penicillins, monobactams, cephalosporins and carbapenems, are antibiotics that act by inhibiting peptidoglycan synthesis. These are molecules used frequently in first-line treatment of serious infections in both city and hospital medicine. Beta-lactamases are enzymes that hydrolyze the beta-lactam nucleus of beta-lactams. This is the most common beta-lactam resistance mechanism. Betalactamases are classified according to Ambler (Ambler, R. P. Philos.Trans, R. Soc.
    [0002] London Ser B, 1980, 289, 321-331) in 4 groups: A: penicillinases, including extended broad-spectrum beta-lactamases ESBL B: metalloenzymes C: cephalosporinases D: oxacillinases In enterobacteria, beta-lactamase production extended-spectrum lactamases (hereinafter referred to as ESBLs) or hyperproduced cephalosporinases is currently the main mechanism of resistance to 3rd generation cephalosporins (hereinafter referred to as C3G), such as cefotaxime, ceftriaxone or ceftazidime, which are oxyimino-cephalosporins. Cefepime, which is the only 4th generation cephalosporin (hereinafter referred to as C4G), is in turn hydrolyzed by ESBLs but usually not by cephalosporinases. Penicillins are also hydrolysed, but not carbapenems. ESBL activity can be inhibited by clavulanic acid, tazobactam or sulbactam (penicillinase inhibitors). The activity of cephalosporinases can be inhibited by cloxacillin. Carbapenemases (enzymes that can belong to classes A, B and D according to Ambler) are enzymes that hydrolyze carbapenems but also most often penicillins and cephalosporins. Accurate and rapid detection of cephalosporin-resistant, ESBL-producing or hyperproduced cephalosporinases, 3034523 2 or carbapenemase-resistant bacteria is crucial for the management of patients. It must lead to the implementation of a suitable antibiotic treatment. In the absence of production of enzymes hydrolysing C3G, the latter can be used in first intention, while in the opposite case a use of 5 carbapenems is necessary. In this case, it is also necessary to determine if these bacteria produce carbapenemases. Heretofore, the conventional technique used to detect bacteria producing beta-lactamases is based on the implementation of an antibiogram and the carrying out of the double synergy test which makes it possible to highlight the restoration of sensitivity to C3G in the presence of penicillinase inhibitor. Despite its effectiveness, the implementation of this technique requires a prior step of culture and isolation of bacteria, which implies a delay of at least 24 hours before the production of beta-lactamases is detectable.
    [0003] The presence of beta-lactamase-producing bacteria also can be demonstrated by molecular biological methods, for example by detecting by PCR and / or sequencing the genes encoding betalactamases. Although they may allow specific characterization of the genes encoding, for example, ESBLs, these laboratory techniques are expensive and time consuming to implement because of the prior extraction of DNA from complex samples. There is therefore an urgent need in the field of health to develop an efficient, rapid and low cost method for detecting the presence of beta-lactamase-producing bacteria in order to detect the presence of beta-lactam-resistant bacterial strains. Nordmann et al. (J.Clin.Microbiol., 2012, 50, 3016-3022) describe a method for detecting ESBL-producing bacteria that involves the use of cefotaxime and a colored pH indicator. This method is based on the fact that the hydrolysis of the beta-lactam nucleus of cefotaxime leads to the formation of a carboxylic acid function and thus to the acidification of the reaction medium. The pH change of the medium is visualized through the use of a colored indicator. Despite its efficiency and speed, this method requires a prior step of binding isolation of bacterial strains.
    [0004] Another colorimetric approach is to use nitrocele, a chromogenic cephalosporin, which can be hydrolysed by a beta-lactamase, thereby causing the color change from yellow to red (O'Callaghan et al., Antimicrob Agents Chemother., 1972, 1, 283-288). Scheme 1 below illustrates the hydrolysis of the beta-lactam nucleus of nitrocele with a beta-lactamase. However, this method again requires the use of previously isolated bacterial strains to enable colorimetric detection. This method can not be used directly on complex samples with staining that may interfere with the colorimetric detection of nitrocele hydrolysis. Here again, this method imposes a delay of at least 24 hours between the taking of a clinical sample and the rendering of the result.
    [0005] In this context, the object of the invention is therefore to provide a method capable of rapidly and effectively determining the presence of beta-lactamase-producing bacteria in a sample without first isolating the bacterial strains.
    [0006] The invention relates to a method for determining in vitro the presence of beta-lactamase-producing bacteria in a sample capable of containing said bacteria, comprising the following steps: (a) incubating said sample in a medium containing a beta-lactamase; lactamine having electrochemical properties, especially a cephalosporin, more particularly nitrocele, as a substrate for beta-lactamases; (b) applying electrochemical analysis means for determining the presence of beta-lactamase producing bacteria. The invention is based on the fact that the hydrolysis of the beta-lactam nucleus of a beta-lactam, particularly the nitrocele, with a beta-lactamase can be detected electrochemically. The inventors have demonstrated for the first time that the electrochemical properties of the hydrolyzed nitrocéfine are distinct from those of the nitrocéfine. Indeed, the hydrolyzed nitrocéfine is an electroactive product capable of generating a specific anodic oxidation current that can be measured by an electrochemical method. The oxidation of the hydrolysed nitrocele produces an anodic current in a range of potentials different from that in which said anode current corresponding to the oxidation of the nitrocele occurs. Thus, the anodic stream specific for the hydrolyzed nitrocele can be used as the analytical response indicating the presence of beta-lactamase-hydrolyzed nitrocaine. In contrast to the methods already developed in the prior art, which require bacteria to be cultured for at least 24 hours beforehand, the method described in the invention makes it possible to detect the presence or absence of producing bacteria. beta-lactamases after only a few hours of incubation of the sample, especially 1 to 4 hours, before the implementation of the electrochemical measurement.
    [0007] The incubation time of a sample with a medium containing the nitrocine is typically 5 to 30 minutes. A cephalosporin-containing medium having electrochemical properties may be a buffered medium in which an electroactive cephalosporin is added. Said buffered medium may be, by way of example, a phosphate buffer of pH 7 and concentration of 100 mM. By "beta-lactamases" is meant a family of enzymes capable of hydrolyzing the 13-lactam nucleus of a 13-lactam. Penicillins, cephalosporins, monobactams and carbapenems are the four families of β-lactams. Depending on their catalytic properties and their affinity for certain substrates as well as their sensitivity to certain types of beta-lactamase inhibitors, the 5 main beta-lactamases are: penicillinases, inducible cephalosporinases, hyperproduced cephalosporinases, spectrum beta-lactamases extended, and carbapenemases. Penicillinases are those enzymes which hydrolyze amino, carboxy and ureidopenicillin. They are inhibited by penicillinase inhibitors clavulanic acid, tazobactam and sulbactam. The term "inducible cephalosporinases" means enzymes that hydrolyze amino-penicillins and cephalosporins of the 1st and / or 2nd generation. They are insensitive to the action of penicillinase inhibitors; however, they are inhibited by cloxacillin. The expression of a gene encoding a cephalosporinase may be induced by an inducer, which may be, in particular, a beta-lactam. The term "hyperproduced cephalosporinases" is understood to mean the enzymes which hydrolyze aminopenicillins, carboxy- and ureidopenicillins, cephalosporins of the 1st, 2nd, 3rd or even 4th generation. They are insensitive to the action of penicillinase inhibitors; however, they are inhibited by cloxacillin. The expression of these enzymes is derepressed and leads to an abundant production of these enzymes. "Extended-spectrum beta-lactamases" (ESBLs) are those enzymes which hydrolyze amino-, carboxy- and ureido-penicillins, cephalosporins of the 1st, 2nd, 3rd and 4th generation and are susceptible to inhibition. penicillinase inhibitors. The term "carbapenemases" is understood to mean enzymes which possess hydrolytic activity on carbapenems but generally also on penicillins and cephalosporins of the 1st, 2nd, 3rd and 4th generation.
    [0008] In accordance with the invention, the beta-lactamases can be produced by Gram-negative bacteria: i) from the family Enterobacteriaceae, in particular the genera Escherichia (in particular Escherichia neck), Klebsiella, Enterobacter, Serratia, Morganella, Proteus, Providencia, Pan toea, Hafnia, Citrobacter, Salmonella, Shigella and Yersinia. Ii) non-fermenting, especially Pseudomonas (in particular P. aeruginosa), Stenotrophomonas and Achromobacter. iii) Acinetobacter (in particular A. baumannii). In a particular embodiment, the aforesaid method of the invention further comprises, before the implementation of step (a), a step for concentrating the bacteria present in a sample capable of containing them. The concentration of the bacteria can be carried out, for example, by filtration or centrifugation. In a particular embodiment, step (a) of the aforesaid method 10 of the invention is carried out in a medium containing the nitrocele. Those skilled in the art will be able to choose an adequate concentration for nitrocine and other substrates or inhibitors. According to the invention, an electrochemical analysis means for carrying out the invention may be a potentiometric measurement, an impedance measurement, a coulometric measurement or an amperometric measurement. In an advantageous embodiment, the electrochemical analysis is carried out by an amperometric measurement. By "amperometric measurement" is meant a measure of the electric current as a function of a potential difference established between the working electrode and the reference electrode. The measurement of the electric current can be carried out by means of known amperometric techniques, preferably by a voltammetry with a potential sweep which can be linear, cyclic, pulse, or alternatively potential jump type, such as chronoamperometry. In a particularly advantageous embodiment of the invention, the presence of the hydrolysed nitrocele is measured by cyclic or linear voltammetry. The implementation of these techniques requires a mounting that can be two or even three electrodes, that is to say a mounting comprising a working electrode, a reference electrode and optionally an auxiliary electrode (against electrode). The working electrode, whose surface serves as a site for electron transfer, may be based on carbon or based on a noble metal or based on a metal oxide. The reference electrode is an electrode 35 whose potential is constant, which makes it possible to impose a precisely defined potential on the working electrode. The reference electrode may be an Ag / AgCl electrode. The counter-electrode, which makes it possible to establish the passage of the electric current with the working electrode, can be manufactured with an inert material, such as platinum or carbon. Those skilled in the art will be able to choose and combine the appropriate electrodes according to their general knowledge. Regarding the method of manufacturing the electrodes, the screen printing technique is preferable, although other industrial manufacturing methods such as rotagravure, inkjet printing or possibly photolithography can be envisaged. The electrodes obtained by screen printing are particularly well suited because they can be mass-produced at low cost, and thus possibly be disposable. In addition, their geometric shape and size can be easily modulated. These electrodes can be screen printed in the form of a sensor and possibly integrated in the bottom of the wells of a microplate or other media or systems for filtering bacterial suspensions and incubation with nitrocele. In a particular embodiment, the amperometric measurement is implemented with a screen-printed sensor. It makes it possible to measure in a small volume of solution of the order of a few microliters.
    [0009] In a particular embodiment, the amperometric measurement is carried out with a device involving three electrodes: an Ag / AgCl reference electrode, a carbon working electrode and a carbon counter-electrode. In another particular embodiment, the amperometric measurement 25 is implemented with a screen-printed sensor comprising an Ag / AgCl reference electrode, a carbon working electrode and a carbon counter-electrode. The presence of the hydrolysed nitrocele is indicated by the presence of an anodic oxidation current in a range of potentials and the absence of said current for a control lacking hydrolyzed nitrocine. When the hydrolyzed nitrocele is measured by cyclic voltammetry, its presence is indicated by a peak of anodic oxidation current specific to the hydrolyzed nitrocele in a determined range of potentials.
    [0010] When the detection is carried out by cyclic voltammetry using an Ag / AgCl reference electrode in a neutral pH buffer containing the nitrocine, the intensity related to the anodic oxidation peak current corresponding to the oxidation of hydrolyzed nitrocele is measured in a range of 5 potentials of between + 0.1 V and +0.5 V, in particular between +0.2 V and +0.4 V, more particularly between +0.23V and +0 , 33V. In particular, when the detection is carried out by cyclic voltammetry in a pH7 phosphate buffer containing the nitrocele, the anodic oxidation peak current bound to the oxidation of hydrolysed nitrocele is at 10 ± 0.3 V. Ag / AgCl. When another type of reference electrode and / or another reaction medium is used to implement the method of the invention, a person skilled in the art will be able to calculate or identify this range of potentials according to his general knowledge.
    [0011] Since the presence of the hydrolysed nitrocele is detected by an electrochemical method, the method described in the invention is particularly advantageous for the analysis of color samples or disorders without prior treatment, with the possibility of offering a quantified measurement with good sensitivity using a low cost apparatus, reduced size, and possibly portable. A sample that can be analyzed by the method of the invention may be a biological sample, an environmental sample or a food sample.
    [0012] A biological sample may be, in particular, a blood sample, a urine sample, a tissue sample or a lung sample. An environmental sample may be wastewater, hospital wastewater, treated wastewater, or sludge from wastewater treatment.
    [0013] In particular, the method of the invention comprises the steps of: (a) incubating a sample capable of containing said bacteria in a medium containing the nitrocine, (b) applying an amperometric measurement respectively to the aforesaid medium obtained at the end of step (a) and a negative control, (c) determine the presence of the beta-lactamase producing bacteria by comparing the value of the anodic current intensity corresponding to the oxidation of the hydrolysed nitrocele measured for the aforesaid medium obtained at the end of step (a), with the value of the intensity of the anode current measured for the negative control. According to the invention, a negative control is a medium containing the nitrocele in the absence of sample.
    [0014] An amperometric measurement applied to said negative control makes it possible to indicate any current that is not related to the oxidation of the hydrolysed nitrocele. In an amperometric measurement, the presence of the hydrolysed nitrocele is indicated by the appearance or increase of an anode current in a range of potentials determined with respect to the measured anode current of the control in the same range. Moreover, the method described in the present invention offers the possibility of quantifying the bacteria producing beta-lactamases in a sample.
    [0015] Indeed, the intensity of the anodic current produced by the oxidation of the hydrolysed nitrocele is quantitatively proportional to the amount of hydrolysed nitrocele. When the amount of nitrocefin is in excess, the amount of hydrolyzed nitrocaine is also proportional to the amount of beta-lactamases involved in the hydrolysis of the nitrocine, as well as to the amount of the bacteria producing these enzymes. Therefore, comparing the value of the anodic current intensity specific to the hydrolyzed nitrocele with a reference value, such as a calibration curve, makes it possible to determine the amount of the beta-lactamase producing bacteria. A particular embodiment of the invention makes it possible both to determine the presence of beta-lactamase producing bacteria in a sample capable of containing them and to quantify them. In this embodiment, the method further comprises, after determining the presence of β-lactamase-producing bacteria, a step of comparing the measured anodic current intensity value for said obtained medium at the end. of step (a) with a calibration curve established under the same conditions. Said calibration curve is established by serially diluting a reference sample containing a known quantity of beta-lactamase producing bacteria and previously determined by any conventional method, such as counting on culture medium or else time PCR. real. In a particular embodiment, the invention relates to a method for the determination and quantification of beta-lactamase producing bacteria in a sample capable of containing said bacteria, said method comprising: (a) incubating a sample capable of containing said bacteria; bacteria in a medium containing the nitrocine, (b) apply an amperometric measurement respectively to the above medium obtained at the end of step (a) and to a negative control, (c) determine the presence of bacteria producing beta-lactamases by comparing the value of the anode current intensity corresponding to the oxidation of the hydrolysed nitrocele measured for the aforesaid medium obtained at the end of step (a), with the value of the intensity of the anodic current measured for 20 the negative control; (d) comparing the value of the intensity of the anodic current measured for the aforesaid medium obtained at the end of step (a) with a calibration curve established under the same conditions for quantifying said bacteria.
    [0016] In another advantageous embodiment, using at least one other beta-lactam and at least one beta-lactamase inhibitor, the method of the invention also makes it possible to discriminate the type of betalactamases. produced by the above-mentioned bacteria in a sample capable of containing them.
    [0017] Indeed, the inventors have found that, in a series of culture media respectively containing a specific substrate and possibly an inhibitor specific to a type of beta-lactamase, the bacteria producing the different types of beta-lactamases can respectively give a profile. Specifically, in terms of anode current intensities for the hydrolyzed nitrocele during the implementation of an amperometric measurement.
    [0018] One of the advantages of this embodiment is that it allows the discrimination of beta-lactamases in complex and colored matrices without prior isolation of the bacteria. More particularly, a method of the invention further makes it possible to discriminate the type of beta-lactamases selected from penicillinases, extended-spectrum beta-lactamases, inducible cephalosporinases, hyperproduced cephalosporinases and carbapenemases, produced by the aforesaid bacteria. in a sample capable of containing them, said method comprising the following steps: (a) incubating in parallel a fraction of said sample in a culture medium A, a culture medium B and a culture medium C: the culture medium A being a basic culture medium, - the culture medium B being a basic culture medium supplemented with a third generation cephalosporin, and the culture medium C being a basic culture medium supplemented with a cephalosporin and a penicillinase inhibitor, and optionally in a culture medium B ', a culture medium C', a culture medium D, a mili The culture medium B 'is a basic culture medium supplemented with a third-generation cephalosporin different from that present in the medium B; the culture medium C 'being the culture medium B' supplemented with a penicillinase inhibitor; the culture medium D being a basic culture medium supplemented by a cephalosporin and a cephalosporinase inhibitor, and the culture medium E being a base culture medium supplemented with a carbapenem, (b) incubating the above-mentioned parallels. culture media obtained at the end of the incubation of step (a) in a medium containing the nitrocele; (C) applying amperometric means to said media obtained at the end of step (b) to determine the presence of beta-lactamase-producing bacteria and to discriminate the type of beta-lactamases. In accordance with the invention, a basic culture medium may be any type of culture medium conventionally used to incubate bacteria, such as LB medium (Luria Bertani broth), TSB (Tryptone soy broth) or BHI (Brain heart infusion). In a particular embodiment, before the implementation of step (b) and after step (a), the aforesaid method of the invention further comprises a step for concentrating the bacteria present in the media. to give a more significant catalytic response. The concentration of the bacteria can be carried out, for example, by filtration or centrifugation. According to the invention, said third generation cephalosporin is selected from cefotaxime, ceftazidime and ceftriaxone; said carbapenem 10 is selected from ertapemem, imipenem or meropenem. According to the invention, said penicillinase inhibitor is in particular chosen from clavulanic acid, tazobactam or sulbactam; said inhibitor of cephalosporinases is in particular cloxacillin. Culture medium B or B 'inhibits the growth of bacteria producing inducible penicillinases or cephalosporinases. Culture medium C or C 'makes it possible to inhibit the growth of bacteria producing a penicillinase or a broad-spectrum beta-lactamase. In a particular embodiment, a culture medium C is a culture medium B supplemented with a penicillinase inhibitor.
    [0019] The culture medium D inhibits the growth of bacteria producing an inducible or hyperproduced cephalosporinase. The culture medium E makes it possible to inhibit the growth of bacteria producing any beta-lactamase except for a carbapenemase. The use of the B, B ', C and C' media makes it possible to discriminate ESBL-producing bacteria which are sensitive to a third-generation cephalosporin, for example cefotaxime, but resistant to another third-generation cephalosporin, for example the ceftazidime, or vice versa. After the respective incubation of the fractions of the same sample in the culture media A, B, C, and optionally the media B ', C', D and E, the beta-lactamases produced by the bacteria from these fractions differentiate themselves. The present invention makes it possible to indicate this difference by an amperometric method which indicates the amount of nitrocele hydrolysed by these beta-lactamases by the values of the intensity of the anodic current corresponding to the oxidation of the hydrolysed nitrocele.
    [0020] The values iA, iE, iE 'ic, ic', ID, and iE are the values of the intensity of the anodic current corresponding to the oxidation of the hydrolyzed nitrocele respectively measured for the bacteria originating from the culture media A, B, B ', C, C', D and E.
    [0021] In the context of the method of the invention, a penicillinase is characterized by the presence of hydrolyzed nitrocele-specific streams obtained for the bacteria originating from a medium A, a medium B and optionally a medium B ' and the absence of the specific current for the bacteria from a medium C and optionally a medium C '. The value iA can be either less than or greater than the value iE and possibly An ESBL is characterized by a high intensity of the anode current specific to the hydrolyzed nitrocele obtained for the bacteria respectively from a medium A, a medium B and optionally a medium B 'and the absence of this specific current for the bacteria from a medium C and optionally a medium C'. The values iE and possibly iE 'are close to the value iA. An inducible cephalosporinase is characterized by an anode stream specific to the hydrolyzed nitrocele obtained for the bacteria from media A, B and optionally B ', C and optionally C', with iA <iE <ic and optionally iA <<and absence of this specific current for the bacteria derived from a medium D. A hyperproduced cephalosporinase is characterized by a high intensity of the anodic current specific to the hydrolysed nitrocele obtained for the bacteria from a medium A, the presence of this specific current. for the bacteria respectively issuing from a medium B and a medium C, optionally a medium B 'and a medium C', with iE> ic and optionally> and the absence of this specific current for the bacteria from a medium D. A carbapenemase is characterized by the presence of an anode stream specific to the hydrolyzed nitrocele obtained for the bacteria from the media A, B and optionally B ', C and optionally C', D and E. In a more particular embodiment, the invention relates to a method for determining the presence of bacteria producing an extended spectrum betalactamase, said method comprising the steps of: 3034523 14 (a) incubating in parallel a fraction of the above sample in a culture medium A, a culture medium B and a culture medium C as defined previously, (b) incubating in parallel the aforementioned culture media obtained at the end of 5 de incubating step (a) in a medium containing the nitrocele, (c) applying an amperometric means to the aforementioned media obtained at the end of step (b) to determine the presence of the bacteria producing an extended spectrum betalactamase .
    [0022] In another more particular embodiment, the invention relates to a method for determining the presence of extended spectrum beta-lactamase producing bacteria, said method comprising the steps of: (a) incubating a portion of said sample in a medium of culture A, a culture medium B, a culture medium B ', a culture medium C and a culture medium C', as defined previously, (b) incubate in parallel the aforementioned culture media obtained at the end of incubation of step (a) in a medium containing the nitrocine, (c) applying amperometric means to the aforesaid media obtained at the end of step (b) to determine the presence of beta-producing bacteria. extended spectrum lactamase. The use of media B, B ', C and C' makes it possible to discriminate ESBL-producing bacteria that are sensitive to a third-generation cephalosporin but resistant to another third-generation cephalosporin. In a particular embodiment, culture medium B is culture medium A supplemented with cefotaxime; the culture medium B 'is the culture medium A supplemented with ceftazidime; the culture medium C is the culture medium B supplemented with a penicillinase inhibitor; the culture medium C 'is the culture medium B' supplemented with a penicillinase inhibitor. In another particular embodiment, culture medium B is culture medium A supplemented with ceftazidime; the culture medium B 'is the culture medium A supplemented with cefotaxime; the culture medium C is the culture medium B supplemented with a penicillinase inhibitor; the culture medium C 'is the culture medium B' supplemented with a penicillinase inhibitor. The use of these media within the scope of the invention makes it possible to discriminate ESBL-producing bacteria that are either cefotaxime-sensitive but resistant to ceftazidime or are cefotaxime-resistant but sensitive to ceftazidime. Advantageously, said method for determining the presence of beta-lactamase-producing bacteria and discriminating the type of beta-lactamases comprises the following steps: (a) incubating a fraction of the above-mentioned sample in a culture medium A, a culture medium B, a culture medium B ', a culture medium C and a culture medium C': 10 - the culture medium A being a basic culture medium, - the culture medium B and the culture medium B ' being basic culture media supplemented respectively by a different third-generation cephalosporin, - culture medium C and culture medium C 'being respectively culture media B and B' supplemented with a penicillinase inhibitor, and optionally in a culture medium D, a culture medium E: - the culture medium D being a basic culture medium supplemented with a cephalosporin and a cephalosporinase inhibitor, and - the mi where E is a basic culture medium supplemented with carbapenem, (b) incubate in parallel the aforesaid culture media obtained at the end of the incubation of step (a) in a medium containing the nitrocele; (c) applying an amperometric means to said media obtained at the end of step (b) to determine the presence of beta-lactamase-producing bacteria and to discriminate the type of beta-lactamases. According to another more particular embodiment, the method of the invention makes it possible to determine and discriminate beta-lactamase-producing bacteria in a sample capable of containing them, said method comprising the steps of: (a) incubating in parallel a fraction of the aforesaid sample in a culture medium A, a culture medium B, a culture medium C, a culture medium D, a culture medium E, optionally a medium B 'and a medium C', as previously defined (B) incubating in parallel the aforesaid culture media obtained at the end of the incubation of step (a) in a medium containing the nitrocine, (c) applying an amperometric measurement respectively to the above said media obtained at the end of step (b) and a negative control, (d) determining the presence of beta-lactamase producing bacteria in said sample by comparing the value of the ano-current intensity. corresponding to the oxidation of hydrolyzed nitrocele obtained for the fraction cultured in culture medium A with the value of the intensity of the current obtained for the negative control, and (e) to discriminate the type of beta-lactamases produced by the said bacteria in said sample, by comparing the respective values of the intensity of the aforesaid anodic current obtained for the fractions cultivated in parallel in the culture media A, B, C, D and E, optionally B 'and C' with the values obtained for a reference bacterial strain.
    [0023] By "reference bacterial strain" is meant a bacterial strain producing a beta-lactamase of the type already identified. In an even more particular embodiment, the method of the invention further comprises a step (f) after step (e) for quantifying bacteria producing the beta-lactamase discriminated in step (e) by comparing the value of the intensity of the anode current corresponding to the nitrocele hydrolysed by said beta-lactamase with a calibration curve established under the same conditions. The value of the anodic current intensity corresponding to the extended-spectrum beta-lactamase hydrolysed nitrocele is calculated according to the formula: IB-ic. The value of the intensity of the anode current corresponding to the nitrocele hydrolysed by a hyperproduced cephalosporinase is calculated according to the formula: IB-ID. The value of the intensity of the anode current corresponding to the carbapenemase-hydrolyzed nitrocrefin corresponds to the value E. More advantageously, the method of the invention can even discriminate the bacteria producing a beta-lactamase subfamily, at the same time. using an additional medium containing a substrate and a beta-lactamase subfamily specific inhibitor.
    [0024] The method of the invention can in particular discriminate the bacteria producing a carbapenemase subfamily, by using an additional medium containing a carbapenem and a specific inhibitor to a subfamily of carbapenemases.
    [0025] The discrimination is based on the specific sensitivity of a carbapenemase subfamily to certain types of inhibitors. For example, metallo-beta lactamases, a subpopulation of Amber class B carbapenemases, are sensitive to EDTA or mercaptoacetic acid, whereas Ambler class A carbapenemases are sensitive to clavulanic acid and boronic acid. According to the invention, a carbapenemase inhibitor is selected from EDTA, mercaptoacetic acid, boronic acid, or clavulanic acid. In another advantageous embodiment, the method of the invention makes it possible to discriminate the type of beta-lactamases and possibly to specify the sub-family of carbapenemases produced by the aforesaid bacteria in a sample capable of containing them, said method comprising the following steps: (a) incubating a fraction of the above sample in parallel: in a culture medium A, a culture medium B and a culture medium C, as defined previously, and optionally in a culture medium B a culture medium C ', a culture medium D, a culture medium E, and a medium of F, the culture media B', C ', D, E being as previously defined, the culture medium F being a basic culture medium supplemented with a carbapenem and an inhibitor specific to a subfamily of carbapenemases; (b) incubating in parallel the aforesaid culture media obtained at the end of step (a) in a medium containing the nitrocine, (c) applying an electrochemical analysis means to the aforesaid media obtained at the end of the step ( b) to determine the presence of beta-lactamase producing bacteria and to discriminate the type of beta-lactamases. The culture medium F makes it possible to inhibit the growth of the bacteria producing a type of carbapenemase which is sensitive to the aforementioned carbapenemase-specific inhibitor. The EDTA and the mercaptoacetic acid are specific inhibitors of the class B then that boronic acid and clavulanic acid are class A specific inhibitors.
    [0026] In a particularly advantageous embodiment, the method of the invention for discriminating the type of beta-lactamases and possibly specifying the subfamily of carbapenemases produced by the aforesaid bacteria comprises the following steps: (a) incubating in parallel a fraction of the aforesaid sample in a culture medium A, a culture medium B, a culture medium C, a culture medium D, a culture medium E, and a culture medium F, as defined previously, and optionally a culture medium B 'and a culture medium C' as defined previously, (b) incubate in parallel the aforesaid culture media obtained at the end of the incubation of step (a) in a medium containing the nitrocele, (c) applying an amperometric measurement respectively to the above media obtained at the end of step (b) and to a negative control, (d) determining the presence of bacteria producing beta-lactamases in s said sample by comparing the value of the intensity of the anode current corresponding to the oxidation of hydrolyzed nitrocele obtained for the fraction cultivated in the culture medium A with the value of the intensity of the current obtained for the negative control, (e) discriminating the type of beta-lactamases produced by the above-mentioned bacteria in said sample by comparing the respective values of the intensity of the aforesaid anodic current obtained for the fractions cultured in parallel in the culture media A, B, C , D, E and F, and optionally the culture media B 'and C', with the respective values obtained for a reference bacterial strain.
    [0027] The method of the present invention can be used during the development of novel 13-lactam antibiotics particularly for the study of the stability of the molecule to different beta-lactams.
    [0028] The present invention is further illustrated by the following figures and examples.
    [0029] FIG. 1: Cyclic voltammograms (y = 50 mV.s -1) obtained for a solution of 500 μM nitrocele in PBS in the absence (dotted line) and in the presence of ESBL (solid line) after 10 min. 'incubation.
    [0030] Figure 2: Log-log of an ESBL whose quantities are determined by the cyclic voltammetric method according to the invention (A, E) and by the spectrophotometric method (B, 0). Analytical responses are recorded after 10 min incubation of ESBLs with a solution of 500 μM nitrocele prepared in PBS. The reaction media are diluted 5-fold in PBS prior to performing the spectrophotometric measurement. The R value corresponds to the current responses or the optical densities, respectively normalized with the values obtained in the absence of ESBL (io = 50 ± 5 nA, ODo = 0.097 ± 0.008). The standard deviation represents the standard error of both measurements. 3: Voltammetric curves (y = 50 mV.s-1) recorded on a screen-printed sensor for E. coli ESBL + (A) and ESBL-B strains, cultured at 37 ° C. for 4h30 in medium of culture containing 4 μg.mL-1 of cefotaxime after centrifugation (1 mL, 7000 g, 10 min) and incubation of the pellet for 10 min with 50 μL of a solution of 500 μM nitrocele in PBS. Figure 4: Cyclic voltammograms (y = 50 mV.s-1) recorded with a screen-printed sensor for E. coli strain ESBL + grown in culture medium containing only 4 μg.mL-1 of cefotaxime (A) or supplemented with 10 μg.mL-1 of potassium clavulanate and then centrifuged (1 mL, 7000 g, 10 min) before a step of incubation of the pellet with 50 μL in a solution of 500 μM nitrocele in PBS for 10 min. .
    [0031] Figure 5: Calibration curve of the ESBL strain + after incubation at 37 ° C for 3h30 in LB culture medium supplemented with 4 μg.mL-1 of cefotaxime. The bacterial culture is then serially diluted and 10 mL volumes are filtered in duplicate. The bacteria recovered after filtration are incubated for 10 min with 80 μl of the 500 μM nitrocrefin solution in PBS. The abscissa axis corresponds to the number of bacteria recovered after filtration. Error bars represent the standard deviation of two experiments. The open square (E) represents the negative control consisting of the ESBL strain + cultured with 4 μg.mL-1 of cefotaxime and 10 μg.mL-1 of clavulanic acid. The open circle (0) corresponds to the signal of the unhydrolyzed nitrocine.
    [0032] Figure 6: Calibration curves of an E. coli strain producing ESBLs in three raw (-) wastewater samples and three pre-autoclaved treated water samples (E). The value i (nA) corresponds to the stream of the oxidation peak of the hydrolysed nitrocele registered for a quantity of bacteria to which was subtracted the one measured for the negative control. Figures 7A, 7B, 7C, 7D, 7E, 7F: Profiles of oxidation current (pA) of the hydrolyzed nitrocaine registered for different types of beta-lactamases and used to discriminate beta-lactamases in medical samples (blood cultures, isolated strains). Examples 1. Materials and methods 15 1.1. Reagents and Solutions The phosphate buffer (PBS, 100 mM, pH = 7.0) and all aqueous solutions are prepared with Milli-Q 18-MQ (Millipore purification system) water. The culture medium, composed of Luria broth, (LB, 10 gL-1 of Bacto tryptone, 5 gL-1 of Bacto yeast extract, 5 gL-1 of NaCl) is prepared in the laboratory and sterilized by autoclaving at 120 ° C. ° C. Nitrocine is purchased from Merck Millipore (Reference 484400). A stock solution of 10-2 M nitrocele is prepared in dimethylsulfoxide (DMSO) and then stored as 50 μL aliquots at -20 ° C.
    [0033] The sodium salt of cefotaxime (Reference C 7039-100mg) and potassium clavulanate (Reference 33454-100mg) are provided by Sigma-Aldrich. Stock solutions at 1 mg.mL-1 are prepared daily in PBS. ESBL was extracted from a strain of E. coli isolated in the Bacteriology laboratory of the University Hospital Center of Dijon (1485, type CTX-M-1).
    [0034] E. coli strain K12 and ESBL producing E. coli strain (MIAE6690) are from the strain collection of INRA Dijon. Samples of these strains are stored at -80 ° C as a 500 μL aliquot containing 50% bacterial suspension and 50% glycerol (v / v). 3034523 21 1.2. Measuring devices and electrodes 1.2.1. Proof of concept The electrochemical measurements are carried out with a PGSTAT12 Autolab (Metrohm) potentiostat controlled by the GPES software (version 4.9).
    [0035] The sensors are prepared with a manual screen printing machine (French Printed Circuit, Bagneux, France) with carbon ink (Electrodag 0 PF 407A, Acheson Colloids). A series of 6 sensors is printed on a flexible polyester film by passing ink through a silkscreen frame (120 threads / cm) using a squeegee. After a drying step (1 h 10 to 65 ° C), the sensors are then stored at room temperature. Each sensor consists of the working electrode and a counter-electrode. The working surface of the sensor (7.07 mm2) is delimited with an adhesive ring that also defines the electrochemical cell. To perform the amperometric measurement, the sensor is inserted into a connector (Dropsens) connected to the potentiostat and then a drop of 20 μL of the solution to be analyzed is deposited on the surface of the sensor. After plunging a silver wire coated with a precipitate of silver chloride (Ag / AgCl) which constitutes the reference electrode, the measurements are made by cyclic voltammetry (y = 50 mV.s-1) at room temperature . 1.2.2 For the discrimination of beta-lactamases in blood samples and the quantification of ESBL-producing strains: The voltammetric measurements (y = 50 mV.s-1) are carried out by depositing drops of 30 μl of solution on screen printed carbon sensors 25 supplied by Dropsens (DRP-110) previously connected to a portable potentiostat PSTAT mini 910 (Methrohm) powered by the USB connection of the computer and controlled by the software PSTAT (version 1.0). 1.3. Amperometric detection of beta-lactamase activity 5 μl of an ESBL solution are introduced into a polypropylene tube containing 45 μl of a solution of 0.5 mM nitrocele in phosphate buffer (PBS, 100 mM; 7.0). After an incubation step of 10 minutes at room temperature in the dark, 20 μl of the mixture are removed and transferred to the surface of the screen-printed carbon sensor. A reference electrode (Ag / AgCl) is then immersed in the previously deposited solution and the cyclic voltammetry measurements are carried out as follows: potential sweep between -0.4 V and 1.2 V at a speed of 50 mV.s-1. The oxidation peak appearing at +0.3 V results from the hydrolysis of the beta-lactam ring of the nitrocele and can be chosen as the analytical response to identify beta-lactamases in a sample. 1.4. Amperometric and Colorimetric Measurement of the ESBL Activity with the Nitrocefin 45 μl of a solution of nitrocele (0.5 mM in PBS) is introduced into a polypropylene tube containing 5 μl of the ESBL solution previously diluted in PBS. The reaction mixture is incubated for 10 min at room temperature in the dark. The product of the enzymatic reaction is then detected by amperometry and spectrophotometry. The electrochemical measurement is carried out by transferring 20 μL of the mixture onto the surface of the screen-printed carbon sensor in order to perform the voltammetric measurement as indicated previously. The intensity of the oxidation peak current measured at + 0.3 V vs. Ag / AgCl is chosen as the analytical answer. For spectrophotometric detection, the solution is diluted in PBS (factor 5) and then pipetted into single-use vats (ratiolab® Q-VETTES semi-micro, no. 2712120) before taking an absorbance measurement at À = 520 nm with a C 800 spectrophotometer (Beckman Coulter). 1.5. Detection of a beta-lactamase-producing strain 10 μl of the non-beta-lactamase producing E. coli K12 strain (BLSE-) and 10 μl of the ESBL-producing E.coli MIAE6690 strain (ESBL) are also grown in parallel. in a tube containing 10 mL of LB medium supplemented with cefotaxime (4 μg.mL-1) for 4h30 at 37 ° C. Then, 30 mL of each bacterial suspension is transferred to a polypropylene tube and centrifuged at 7000 g for 10 min. After removing the supernatant, a volume of 50 μl of a solution of nitrocaine (0.5 mM in PBS) is introduced into the tube and incubated with the bacterial pellet for 10 min at room temperature in the dark. The liquid is then sucked and transferred to the screen-printed sensor in order to perform the voltammetric measurement as indicated in section 1.3. 1.6. Identification of strains resistant to 3rd generation cephalosporins in blood cultures The blood cultures supplied by the Bacteriology laboratory of the Dijon Regional Hospital are blood samples which have been cultured either in Bactec ™ vials suitable for the search for mainly aerobic microorganisms. either in BactecTM vials dedicated to the growth of mainly anaerobic bacteria. 10 .mu.l volumes of sample are in parallel introduced into tubes containing 10 ml of LB medium (Medium A), 10 ml of LB medium supplemented with cefotaxime at 4 .mu.g -ML (Medium B) and 10 ml of medium. LB 15 supplemented with cefotaxime at 4 μg.mL-1 and clavulanic acid at 100 μg.mL-1 (Medium C). After an incubation step at 37 ° C. with stirring for 2 hours, 5 ml of the contents of each tube are taken with a syringe and then filtered using a manual filtration device (Swinnex0, 13 mm, Millipore) equipped a 0.45 μm porosity membrane (HVLP, 13 mm, Millipore). Each filter is then placed at the bottom of a polystyrene microwell (24-well plate, Cellstar ™, 662160) into which 80 μL of a solution of nitrocaine (0.5 mM in PBS) is then added, and then left behind. Incubate for 10 min at room temperature in the dark. pL of each mixture are taken and transferred to the surface of a screen-printed carbon sensor (Dropsens, DRP-110) previously connected to a PSTAT mini 910 portable potentiostat (Methrohm) powered by the USB connection of the computer and driven by PSTAT software (version 1.0). Linear voltammetric measurements are made by performing a linear potential scan from -0.1 V up to 1.2 V at a speed of 50 mV.s-1.
    [0036] The peak current (i) appearing at + 0.3 V, related to the hydrolysis of the beta-lactam ring of the nitrocine, is chosen as the analytical response. Comparison of the current values i measured for each incubation condition of the sample (medium A, B, C) makes it possible to conclude on the capacity of the strain to produce or not a hydrolyzing enzyme cefotaxime. 3034523 24 1.7. Quantification of extended spectrum beta-lactamase producing strains (ESBL) in wastewater 1.7.1. Production of an ESBL-Producing E. coli Calibration Curve in Sewage Treatment Waters 10 μl of the E.coli MIAE6690 strain producing ESBL are cultured in a tube containing 10 ml of culture medium. LB supplemented with cefotaxime (4 μg.mL-1) overnight at 42 ° C with shaking. The bacterial concentration in the so obtained culture (So) is determined by serial dilutions in TS medium containing Tryptone (10 gL-1) and NaCl (5 μL-1) and then spreading the solutions. on plates containing agar medium (35.6 gL-1Tryptone Bile X-glucose, 4 mg.L-1 cefotaxime). The So culture is serially diluted (dilution factors between 10 and 108) in a sample of scrubber water (either crude or processed) pre-autoclaved by preparing 1 mL solution volumes in tubes. made of polypropylene. Then, 500 μl of these previously diluted solutions (dilution factors between 104 and 108) are separately introduced into a tube containing 25 ml of LB medium supplemented with cefotaxime at 4 μg.mr1 and incubated at 42 ° C. with shaking for 4 hours. A volume of 500 μL of the sample of water treatment plant (either raw or treated) pre-autoclave, used to make the dilutions of the bacterial strain, is incubated under the same conditions (negative control). 10 ml volumes are then collected in duplicate in each tube with a syringe and then filtered separately using a manual filtration device (Swinnex ™, 13 mm, Millipore) equipped with a 0.45 μm porosity membrane. (HVLP, 13 mm, Millipore). Each filter is then placed at the bottom of a polystyrene microwell (24-well plate, Cellstar®, Reference: 662160) into which are then introduced 80 μl of a solution of nitrocele (0.5 mM in PBS), then left incubate for 15 minutes at room temperature in the dark. 40 μl of the contents of each well are taken and transferred to the surface of a screen-printed carbon sensor (Dropsens, DRP-110) previously connected to a PSTAT mini 910 portable potentiostat (Methrohm) and the linear voltammetry measurements are carried out as mentioned in part 1.6. 1.7.2. Detection of ESBL-Producing Strains in Sewage Water Samples Sewage (1 - 10 mL) and treated water (20 - 100 mL) volumes are screened in duplicate on porosity membranes 0.45 μm (HAWP, 47 mm, Millipore) using a stainless steel filtration boom (Sartorius Combisart). For each sample, one of the membranes is introduced into a tube containing 25 ml of LB medium supplemented with cefotaxime at 4 μg.mL-1 (Medium B) while the second is dipped into a tube containing 25 ml of supplemented LB 10 medium. with cefotaxime at 4 μg.mL-1 and clavulanic acid at 10 μg.mL-1 (Medium C). All the tubes are incubated at 42 ° C. with stirring for 4 hours, then their contents are filtered and analyzed in the presence of nitrocine as indicated in 1.7.1. For a given sample, the intensities of the peak currents measured at 0.3 V for the incubations in the media B and C are respectively denoted iB and ic. The amount of ESBL producing strains is calculated from the current value i = iB - 'ce using a calibration curve (1.7.1). 2. Results 20 2.1. Electrochemical characterization of the hydrolysed nitrocele The nitroefine was hydrolysed by extended-spectrum beta-lactamase in abundant quantities. The voltammetric behavior of nitrocele (S) and its hydrolysed form (P) were studied by cyclic voltammetry using screen - printed electrodes based on carbon. Both the nitrocrefin and the hydrolyzed nitrocrefin exhibit an irreversible anodic oxidation peak (α1) at about +1.0 V. Ag / AgCl. This peak can be attributed to the oxidation of the thiol group in the dihydrothiazine group or to other functions specific to cephalosporin derivatives. In contrast to nitrocine, the hydrolyzed nitrocele (P) generates a well-defined irreversible anodic oxidation peak (a2) and observed at a lower anodic potential of about +0.3 V (FIG. The presence of the peak a2 indicates that of the hydrolysed nitrocele and can therefore be chosen as the analytical response to demonstrate the activity of the extended spectrum betalactamase. 2.2. Measurement of ESBL Activity The activity of ESBLs was measured in parallel by spectrophotometric measurement and voltammetric measurement using nitrocaine. Each series of experiments is performed by varying the ESBL concentration and the log-log curves obtained with the electrochemical method of the invention and the spectrophotometric method are illustrated in FIG. 2. Each measurement is normalized compared to the signal obtained in the absence of ESBL. The calibration curve obtained by the voltammetric method shows that this method allows a quantitative detection of ESBL activity with a sensitivity close to that of the spectrophotometric method. In addition, the voltammetric method of the invention provides a wider linearity zone. The reproducibility of the spectrophotometric measurement and that of the voltammetric measurement are similar. 2.3. Detection of ESBL producing E. coli strains by measurement of cyclic voltammetry In order to evaluate the ability of the voltammetric method to detect ESBL-producing bacteria, two E. coli strains with a well-characterized genotype were selected. one producing one ESBL (ESBL) and the other not producing ESBL (ESBL). Both strains are first cultured in LB medium containing 4 μg.mL-1 of cefotaxime. After the centrifugation step, the respective bacterial pellets of these two strains are incubated with the nitrocele as a substrate. Figure 3 shows the voltammetric responses respectively obtained for the strain ESBL ± (curve A) and the strain BLSE- (curve B). Curve A has an anodic peak at about +0.4 V vs.. Ag / AgCl, associated with the catalytic hydrolysis of the 13-lactam nucleus of the nitrocele by the ESBL strain, while no peak is recorded for the strain BLSE-. This result agrees well with the optical density values of 0.934 and 0.002 respectively obtained for the strain ESBL ± and the strain BLSE-. The culture medium supplemented with cefotaxime and the antibiotic-free medium were also subjected to voltammetric measurement. No specific signal is observed in the range of potentials [0.1-0.6 V vs. Ag / AgCl] for these two media, which confirms that neither LB culture medium nor cefotaxime interferes with voltammetric detection. 2.4 2.4. Discrimination of ESBL-producing bacteria Since hyperpermeate cephalosporinases and carbapenemases can also hydrolyze cefotaxime, the bacteria producing these enzymes can also give a positive response in the presence of cefotaxime. In order to distinguish between extended-spectrum beta-lactamase producing bacteria and hyperproduced cephalosporinase producing bacteria and carbapenemases, the bacteria were cultured simultaneously in two culture media, one containing cefotaxime as an antibiotic, the other containing in addition potassium clavulanate as an inhibitor of ESBL. After the recovery of the bacteria, the bacterial pellets were incubated with the nitrocaine as substrate. The voltammetric curves recorded by cyclic voltammetry are shown in FIG. 4. The anode peak resulting from the hydrolysis of the nitrocele was observed for an ESBL strain grown with cefotaxime (curve A), whereas no signal was recorded for the same strain grown in the presence of potassium clavulanate (curve 15 B). This result confirms the presence of ESBL-producing bacteria. This result is in agreement with the values obtained for the enumeration of E. coli on a selective medium since these are respectively 80 cfu.mL-1 for a culture supplemented with an inhibitor and 1.8 x 108 cfu.mL. -1 for a culture without inhibitor. 2.5. Quantification of bacteria producing beta-lactamases The capacity of the method of the invention for a quantitative determination of ESBL-producing bacteria was evaluated by a series of dilution of a culture of ESBL E.coli ± 5 x 104 to 5 x 10 7 cfu.mL-1, incubated with cefotaxime. The calibration curve of FIG. 5 has a broad zone of linearity which makes it possible to envisage the quantitative determination of bacterial strains producing ESBLs. The plateau observed at high concentrations of ESBL-producing bacteria suggests that all of the initially present nitrocaine is hydrolyzed. 2.6. Quantification of beta-lactamase producing bacteria in raw or autoclaved wastewater ESBL-producing E. coli calibration curves are established for raw wastewater samples and pre-autoclaved treated water samples according to method described in section 1.7.1. These calibration curves are used to quantify the ESBL-producing bacteria respectively in the raw water and the treated water according to the method of the invention. As shown in Tables 1 and 2, the results obtained with a method of the invention are consistent with the results obtained by counting bacteria. Table 1: Determination of ESBL producing strains (ufc.L-1) in raw water from five Côte d'Or treatment plants (A, B, C, D, E). Enumeration Method according to the invention A 2 x 105 1.2 x 105 B 7 x 105 6 x 105 C 1 x 105 2.2 x 105 D 8 x 105 5 x 105 E 3.5 x 105 4 x 105 Table 2 : Determination of ESBL-producing strains (ufc.L-1) in treated water from five Côte d'Or treatment plants (A, B, C, D, E). Enumeration Method according to the invention A 2.5 x 103 1 x 103 B 1 x 104 2 x 104 C 4 x 103 4 x 103 D 1.8 x 103 5 x 103 E 5 x 104 4 x 104 2.7. Discrimination of beta-lactamases Five bacterial strains derived from clinical samples and producing different types of beta-lactamases (penicillinases, CTX-M ESBL, inducible cephalosporinase, hyperproduced cephalosporinase) were analyzed by the method of the invention. invention. Each bacterial strain, as well as the negative control, were respectively incubated in three different culture media (media A, B and C): medium A being an LB culture medium, medium B being a LB culture medium containing 4 pg.mL-1 of cefotaxime, medium C being a LB culture medium containing 4 μg.mL-1 cefotaxime and 100 μg.mL-1 clavulanic acid. After filtration of the bacterial suspensions with SwinnexC), the filtration membranes on which the bacteria were recovered were incubated in the dark for 10 min with 80 μL of 500 μM nitrocele. The measurement of cyclic voltammetry was carried out using carbon electrodes. The anodic current peaks corresponding to the oxidation of the hydrolysed nitrocele for these three media are respectively denoted iA, iB and ic. The results obtained with the producing strains of the various beta-lactamases are collated in FIGS. 7A to 7F.
    [0037] The results in Figure 7A correspond to the response of a sample that contains a strain that does not produce beta-lactamases (negative). The results of Figures 7B and 7C correspond to two penicillinases. The catalytic activity of a penicillinase is characterized by the presence of the anodic stream of the hydrolysed nitrocele obtained for the bacteria from media A and B and the absence of the specific anodic current for the bacteria from medium C. An inducible cephalosporinase (Figure 7D) is characterized by the peaks of the anodic current of the hydrolyzed nitrocele obtained in these three media corresponding to the following criteria: iA <1, iA <iB <ic, and ic> 1.
    [0038] CTX-M extended-spectrum beta-lactamase (FIG. 7E) is characterized by the peaks of the anodic current of the hydrolysed nitrocele obtained in these three media corresponding to the following criteria: iA> 3, iB> 3, and ic < 0.2.
    [0039] A hyperproduced cephalosporinase (FIG. 7F) is characterized by the peaks of the anodic current of the hydrolysed nitrocele obtained in these three media corresponding to the following criteria: iA> 3, iA> iB> ic, and ic> 1. An agar diffusion antibiogram and the amperometric method of the invention are respectively used to discriminate beta-lactamase producing bacteria in blood culture samples. As shown by the results summarized in Table 3 below, the recovery rate of the method of the present invention is 100% with the antibiogram.
    [0040] Table 3: Comparison of the results obtained after the performance of an antibiogram (ATB) and the implementation of the electrochemical method of the invention for the discrimination of beta-lactamase-producing strains in blood culture broths. ATB Electrochemistry Sample 035 Escherichia coli C3G and others: S iA = 0.15 - iB = 0.13 - ic = 0.15 No beta-lactamase C3G and others: S 034 Escherichia C3G: R ESBL) iA = 5.1 - iB = 3.1 - ic = 0.17 blue SS C3G: R 033 Escherichia C3G: R (ESBL) iA = 3.7 - iB = 3.1 - ic = 0.18 deci ESBL C3G: R 030 Proteus C3G: S iA = 2 - iB = 0.3 - ic = 0.18 mira bilis (Penicillinase) Penicillinase C3G: S 029 Escherichia coli C3G: S iA = 1.3 - iB = 2, 2- ic = 0.16 (Penicillinase) Penicillinase C3G: S 026 Escherichia C3G and others: S iA = 0.16 - iB = 0.14 - ic = 0.14 3034523 31 neck i (no beta) No beta lactamase lactamase) C3G and others: S 025 Escherichia coli C3G: S iA = 1.4 - iB = 1.5 - ic = 0.2 (Penicillinase) Penicillinase C3G: S 024 Escherichia coli C3G: S iA = 0 , 7 - iB = 1.5 - ic = 0.15 (Penicillinase) Penicillinase C3G: S 019 Escherichia coli C3G and others: S iA = 0.17 - iB = 0.17 - ic = 0.16 No beta -Lactamase C3G and others: S 005 Esch erichia neck C3G: S iA = 0.42 - iB = 0.96 - ic = 0.18 (Penicillinase) Penicillinase C3G: S * iA, iB, and ic (pA) An agar diffusion susceptibility and amperometric method of the invention are also used to discriminate beta-lactamase producing bacteria from various biological samples, after their isolation on Drigalski lactose agar. The results summarized in Table 4 indicate that the recovery rate of the method of the present invention is 100% with the antibiogram.
    [0041] Table 4: Comparison of the results obtained after the performance of an antibiogram (ATB) and the implementation of the electrochemical method of the invention for the discrimination of previously isolated beta-lactamase producing strains on Drigalski medium. No. ATB Flora Electrochemistry * Sample 018 Enterobacter C3G: S iA = 0.23 - iB = 0.27-ic = 1.3 cloacae cephalosporinase Cephalosporinase inducible inducible C3G: S 3034523 32 014 Escherichia coli C3G: R (ESBL) iA = 5.5 - iB = 5.6 - ic: 0.23 ESBL BSE: R 013 Escherichia coli C3G: R (ESBL) iA = 5.8 - iB = 5.6 - ic = 0.13 C3G ESBL: R 012 Escherichia coli C3G: R (ESBL) iA = 5.0- iB = 3.1 - ic = 0.19 ESBL ESG: R 008 Klebsiella C3G: S iA = 0.25 - iB = 0.55 - ic = 0.24 oxytoca (urine) (Penicillinase) Penicillinase C3G: S 007 Proteus mirabilis (bone) C3G: S iA = 0.14 - iB = 1.3- ic = 0.16 (Penicillinase) Penicillinase C3G: S004 Proteus mirabilis (pus) C3G: S iA = 2.8 - iB = 3.4 - ic = 0.4 (Penicillinase) Penicillinase C3G: S * iA, iB, and ic (pA)
    权利要求:
    Claims (12)
    [0001]
    REVENDICATIONS1. A method for determining in vitro the presence of beta-lactamase producing bacteria in a sample that may contain said bacteria, said method comprising the steps of: (a) incubating said sample in a medium containing a beta-lactam having electrochemical properties, including a cephalosporin, particularly nitrocele, as a substrate for beta-lactamases, (b) applying electrochemical analysis means to determine the presence of beta-lactamase producing bacteria.
    [0002]
    2. Method according to claim 1, characterized in that said electrochemical analysis means is a potentiometric measurement, an impedancemetry measurement, a coulometric measurement or an amperometric measurement. 15
    [0003]
    3. Method according to claim 2, characterized in that said method comprising the following steps: (a) incubating a sample capable of containing said bacteria in a medium containing the nitrocele, (b) applying an amperometric measurement respectively to said medium obtained at the end of step (a) and at a negative control, (c) determine the presence of beta-lactamase producing bacteria by comparing the value of the anode current intensity corresponding to the oxidation of nitrocaine hydrolyzed measured for the aforesaid medium obtained at the end of step (a), with the value of the intensity of the anode current measured for the negative control.
    [0004]
    4. A method according to claim 3 for determining and quantifying beta-lactamase producing bacteria in a sample capable of containing said bacteria, said method further comprising, after step (c), a step (d) of comparing the value of the intensity of the anode current measured for said medium obtained at the end of step (a) with a calibration curve established under the same conditions. 3034523 34
    [0005]
    5. The method of claim 1 or 2 further allowing to discriminate the type of beta-lactamases selected from penicillinases, extended-spectrum beta-lactamases, inducible cephalosporinases, hyperproduced cephalosporinases and carbapenemases, produced by the aforesaid bacteria in a sample capable of containing them, said method comprising the following steps: (a) incubating in parallel a fraction of said sample in: a culture medium A, a culture medium B and a culture medium C: the culture medium A being a basic culture medium, - culture medium B being a basic culture medium supplemented with a third generation cephalosporin, and - culture medium C being a basic culture medium supplemented with a cephalosporin and an inhibitor penicillinases, and optionally in a culture medium B ', a culture medium C', a culture medium D, a culture medium E: the culture medium B 'being a basic culture medium supplemented with a third generation cephalosporin different from that present in the medium B; the culture medium C 'being the culture medium B' supplemented with a penicillinase inhibitor; the culture medium D being a basic culture medium supplemented with a cephalosporin and a cephalosporinase inhibitor, and the culture medium E being a base culture medium supplemented with a carbapenem, (b) incubating the aforementioned media in parallel; culture obtained at the end of the incubation of step (a) in a medium containing the nitrocele; (c) applying amperometric means to said media obtained at the end of step (b) to determine the presence of beta-lactamase-producing bacteria and to discriminate the type of beta-lactamases.
    [0006]
    The method of claim 5 for determining the presence of extended spectrum beta-lactamase producing bacteria, said method comprising the steps of: (a) incubating a fraction of said sample in a culture medium A, a culture medium B and a culture medium C, and optionally in a culture medium B 'and a culture medium C', as defined according to claim 5, (b) incubate in parallel the aforesaid culture media obtained at the end of incubation of step (a) in a medium containing the nitrocine, (c) applying amperometric means to the aforesaid media obtained at the end of step (b) to determine the presence of the beta-lactamase producing bacteria. extended spectrum.
    [0007]
    The method of claim 5 for determining and discriminating beta-lactamase producing bacteria in a sample capable of containing them, comprising the steps of: (a) incubating a portion of said sample in a culture medium A in parallel, culture medium B, culture medium C, culture medium D, culture medium E, optionally medium B 'and medium C', as defined in claim 5; said culture media obtained at the end of the incubation of step (a) in a medium containing the nitrocine, (c) applying an amperometric measurement respectively to the aforesaid media obtained at the end of step (b) and to a negative control, (d) determining the presence of beta-lactamase producing bacteria in said sample by comparing the value of the anodic current intensity corresponding to the oxidation of nitrocele hydrolys e obtained for the fraction cultured in the culture medium A with the value of the intensity of the current obtained for the negative control, and (e) discriminating the type of beta-lactamases produced by the said bacteria in said sample, by the comparing the respective values of the intensity of the aforesaid anodic current obtained for the fractions cultivated in parallel in the culture media A, B, C, D and E, optionally B 'and C' with the respective values obtained for a reference bacterial strain . 30
    [0008]
    The method of claim 7, further comprising a step (f) after step (e) for quantifying bacteria producing the beta-lactamase discriminated in step (e) by comparing the intensity value. the anode current corresponding to the nitrocele hydrolysed by said beta-lactamase with a calibration curve established under the same conditions. 3034523 36
    [0009]
    9. Method according to any one of claims 5, 7-8, for discriminating the type of beta-lactamases and optionally specifying the sub-family of carbapenemases produced by the aforesaid bacteria in a sample 5 may contain them, said method comprising the following steps: (a) incubating a fraction of the above sample in a culture medium A, a culture medium B, a culture medium C, a culture medium D, an culture medium E, and a culture medium F, and optionally a culture medium B 'and a culture medium C', the media A, B, B ', C, C', D, E being as defined in claim 5, the culture medium F being a basic culture medium supplemented with a carbapenem and an inhibitor specific to a subfamily of carbapenemases; (b) incubating in parallel the aforesaid culture media obtained at the end of the incubation of step (a) in a medium containing the nitrocine, (c) applying an amperometric measurement respectively to the aforesaid media obtained at the end of the step (b) and a negative control, (d) determining the presence of beta-lactamase producing bacteria in said sample by comparing the value of the anodic current intensity corresponding to the oxidation of the hydrolysed nitrocaine obtained for the fraction cultured in culture medium A with the value of the intensity of the current obtained for the negative control, (e) discriminating the type of beta-lactamases produced by the above-mentioned bacteria in said sample, by comparing the values of the intensity of the aforesaid anodic current obtained for the fractions cultivated in parallel in the culture media A, B, C, D, E and F, and possibly the culture media. ure B 'and C', with the respective values obtained for a reference bacterial strain. 30
    [0010]
    10. Method according to any one of claims 3 to 9, characterized in that the intensity of the anode current corresponding to the oxidation of the hydrolysed nitrocele in a neutral pH buffer is measured in a range of potentials between + 0 , 1 V and + 0.5 V vs. Ag / AgCl, in particular between + 0.2 V and + 0.4 V vs. Ag / AgCl, more particularly between + 0.23V and + 0.33V vs. Ag / AgCl. 3034523 37
    [0011]
    11. Method according to any one of claims 5 to 9, characterized in that a third generation cephalosporin is selected from the group comprising cefotaxime, ceftazidime and ceftriaxone; that a penicillinase inhibitor is clavulanic acid, tazobactam or sulbactam; said cephalosporinase inhibitor is cloxacillin; that carbapenem is ertapemem, imipenem or meropenem; a carbapenemase inhibitor is selected from EDTA, mercaptoacetic acid, boronic acid, or clavulanic acid. 10
    [0012]
    12. Method according to any one of claims 1 to 11, characterized in that said sample is selected from a biological sample, a sample of environmental origin, or a food sample. 15
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    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO2009051838A1|2007-10-19|2009-04-23|Becton Dickinson And Company|Methods and compositions for the detection of beta-lactamases|
    US20130089883A1|2010-03-01|2013-04-11|Bio-Rad Innovations|Quick method for detecting enyzmes and microoranisms|
    WO2013072494A1|2011-11-18|2013-05-23|INSERM |Method for detecting the presence of expanded spectrum b-lactamase-producing bacteria in a sample|
    US5641623A|1995-01-04|1997-06-24|Martin; Mark T.|Electrochemiluminescence assay|
    CN1729299A|2002-10-29|2006-02-01|昭和药品化工株式会社|Reagent composition for detecting beta-lactamase, detection kit and detection method|
    DE10311315A1|2003-03-14|2004-09-30|Apibio Sas|Method and device for the detection of biomolecules|
    US20050069901A1|2003-09-29|2005-03-31|Eppendorf Ag|Method for detecting microbial antibiotic resistance|
    US9340819B2|2011-06-22|2016-05-17|INSERM |Method for detecting the presence of carbapenemase-producing bacteria in a sample|
    EP3158339A1|2014-06-19|2017-04-26|Université Catholique de Louvain|Method for detecting enzyme activity hydrolyzing beta-lactam ring antimicrobial agents|JPWO2018181997A1|2017-03-31|2020-02-06|国立大学法人大阪大学|Method for detecting carbapenemase producing bacteria|
    WO2020254711A1|2019-06-21|2020-12-24|Servicio Andaluz De Salud|Kit and method for rapid detection of bacteria resistant to combinations of β-lactam antibiotic with a β-lactamase inhibitor, and for rapid detection of extended-spectrum resistance to β-lactams in clinical isolates|
    CN113249434A|2021-05-07|2021-08-13|山西大学|Method for simultaneously detecting bacteria producing extended-spectrum beta-lactamase and carbapenemase|
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    优先权:
    申请号 | 申请日 | 专利标题
    FR1552928A|FR3034523B1|2015-04-03|2015-04-03|NOVEL METHOD FOR DETECTING THE PRESENCE OF BACTERIA PRODUCING BETA-LACTAMASES.|
    FR1552928|2015-04-03|FR1552928A| FR3034523B1|2015-04-03|2015-04-03|NOVEL METHOD FOR DETECTING THE PRESENCE OF BACTERIA PRODUCING BETA-LACTAMASES.|
    CN201680031188.3A| CN107660233B|2015-04-03|2016-04-01|Novel method for detecting the presence of beta-lactamase producing bacteria|
    JP2018502333A| JP6748701B2|2015-04-03|2016-04-01|A novel method for detecting the presence of beta-lactamase-producing bacteria|
    EP16717283.2A| EP3278095B1|2015-04-03|2016-04-01|Novel method for detecting the presence of beta-lactamase-producing bacteria|
    BR112017021186-6A| BR112017021186B1|2015-04-03|2016-04-01|method for detecting the presence of beta-lactamase-producing bacteria|
    US15/562,845| US10718006B2|2015-04-03|2016-04-01|Methods for detecting beta-lactamase-producing bacteria|
    PCT/EP2016/057284| WO2016156605A1|2015-04-03|2016-04-01|Novel method for detecting the presence of beta-lactamase-producing bacteria|
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