RU2776822C2 - Detection of enterobacteriales, pseudomonas and acinetobacter species producing carbapenemase, using chromogen - Google Patents

Abstract

FIELD: microbiology.

SUBSTANCE: invention relates to the detection of the presence of Enterobacteriales, Pseudomonas and Acinetobacter producing carbapenemase in a biological sample. A method is proposed for the detection of the presence of Enterobacteriales, Pseudomonas and Acinetobacter producing carbapenemase in a biological sample, including provision of a sample, in which the presence of Enterobacteriales, Pseudomonas and Acinetobacter producing carbapenemase is supposed, reaction of the specified sample with a solution of (7R)-7-[(z)-2-(2-aminothiazole-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dinitrostyryl)-3-cefem-4-carboxylic acid) or its salt. In this case, the reaction is carried out in the presence of an inhibitor of extended-spectrum β-lactamase (ESBL), AmpC inhibitor, or a mixture thereof. A change of color from yellow to orange/red in a reaction medium is detected, when Enterobacteriales, Pseudomonas and Acinetobacter producing carbapenemase are present in the studied sample. At the same time, change of color occurs during 1 h of sample reaction. The use of (7R)-7-[(z)-2-(2-aminothiazole-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dinitrostyryl)-3-cefem-4-carboxylic acid) or its salt for the detection of the presence of Enterobacteriales, Pseudomonas and Acinetobacter producing carbapenemase in a sample, and a set for the determination of whether a microorganism produces carbapenem-hydrolyzing β-lactamase, containing (7R)-7-[(z)-2-(2-aminothiazole-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dinitrostyryl)-3-cefem-4-carboxylic acid) or its salt, are also proposed.

EFFECT: invention provides for high sensitivity and specificity.

15 cl, 4 dwg, 5 tbl, 4 ex

Description

Technical field

The present invention relates to a method for detecting the presence of carbapenemase-producing Enterobacteriales, Pseudomonas and Acinetobacter in a sample, and to kits for use in such methods.

State of the art

Carbapenemases are a group of β-lactamases that are active against various antibiotics, including carbapenems. Carbapenemases belong to three classes of beta-lactamases, namely Ambler class A, Ambler class B and Ambler class D. These three classes of carbapenemase confer resistance to carbapenems or reduced sensitivity to carbapenems in clinically important bacteria. Thus, bacteria that produce carbapenemase are of clinical interest, and methods for their early detection are required in order to prevent their spread and reduce multidrug resistance and all drug resistance.

Two tests are widely used in clinical practice to detect carbapenemase-producing bacteria: "Etest®" and other products in the form of text strips to determine the MIC (minimum inhibitory concentration) and the "Modified Hodge test" (Modified Hodge-Test). In the Etest® test, the indicator carbapenem and carbapenem, together with antimicrobial enzyme inhibitors active against the microorganism under test, are provided as oppositely directed predefined gradients on agar and are used to determine the minimum inhibitory concentration (MIC) of the antimicrobial agent and the ratio of inhibitory concentrations. In the "modified Hodge test", carbapenemase-producing bacteria are detected when the test bacterium grows in the direction of the carbapenem-containing disk, forming a characteristic "cloverleaf" pattern of growth around the disk. This phenomenon may be mediated by carbapenem-inactivating enzymes produced by the isolate under study. Molecular detection techniques for carbapenemase genes have also been applied. However, all three tests have their drawbacks. "Etest®" and "modified Hodge test" are neither sufficiently specific nor sensitive enough, while molecular detection techniques are complex and expensive. Moreover, all three detection methods are very time consuming, with typical times for determining whether a sample contains carbapenemase-producing bacteria up to 24 hours. This is unsatisfactory in the case of control of nosocomial infections and the transfer of drug resistance.

Acido-colorimetric techniques have recently been developed for the detection of carbapenemase-producing bacteria, which are reported to significantly reduce the time of analysis compared to "Etest®", "modified Hodge test" and molecular detection techniques. International application WO 2012/175637 describes such a method, which, in particular, reports the timing of the analysis, which is less than 2 hours. However, current rapid phenotypic tests show poor sensitivity and specificity for detecting carbapenemase-producing Pseudomonas (Heinrichs et at., 2015) and Acinetobacter in a sample. In particular, false negative signals can be obtained.

In addition, the rapid detection of carbapenemase-producing Enterobacteriaceae is key to limiting the spread of these organisms, and although both phenotypic and molecular methods are available for their detection, no single detection method has proven to be ideal for all situations (Lutgring et al. Limbago, 2016). According to recent taxonomic studies by the European Committee on Antimicrobial Susceptibility Testing (EUCAST, 2018), the definition of the Enterobacteriaceae family has been narrowed. Therefore, the Enterobacteriales are an order of Gram-negative bacteria that includes only one family, the Enterobacteriaceae. Thus, the present invention will refer to the order Enterobacteriales.

Therefore, there is a need to provide a method for detecting the presence of carbapenemase-producing Enterobacteriales, Pseudomonas, and Acinetobacter in a sample that is not only highly specific and sensitive, but can also be easily applied in a clinical setting.

The essence of the invention

The present inventors have found that when performing a phenotypic test using a chromogenic cephalosporin, (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3 -(2,4-dinitrostyril)-3-cephem-4-carboxylic acid or a salt thereof, it is possible to detect the presence of carbapenemase-producing Enterobacteriales, Pseudomonas and Acinetobacter in a very short time.

In particular, the detection process established by the present inventors does not require isolates to be previously exposed to any agents that can induce or inhibit expression prior to testing for the presence of carbapenemase, thus requiring an additional information period.

In one aspect, the present inventors have found that it is possible to detect the presence of carbapenemase-producing Enterobacteriales, Pseudomonas, and Acinetobacter in samples from patients suspected of being infected with such bacteria using a chromogenic cephalosporin, (7R)-7-[(z)-2 -(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dinitrostyryl)-3-cephem-4-carboxylic acid or a salt thereof.

Thus, according to a first aspect, the present invention relates to a method for detecting the presence of carbapenemase-producing Enterobacteriales, Pseudomonas, and Acinetobacter in a sample from a patient, comprising: providing a sample in which the presence of carbapenemase-producing Enterobacteriales, Pseudomonas, or Acinetobacter is suspected; providing a reaction of the sample with (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dinitrostyryl)-3-cephem-4 solution α-carboxylic acid or its salt and detecting a color change in the reaction medium, wherein the color change from yellow to red indicates the presence of carbapenemase-producing Enterobacteriales, Pseudomonas or Acinetobacter in the test sample.

According to a second aspect, the present invention relates to the use of (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2.4 -dinitrostyril)-3-cephem-4-carboxylic acid or its salt to detect the presence of carbapenemase-producing Enterobacteriales, Pseudomonas and Acinetobacter in the sample.

According to a third aspect, the present invention also relates to a kit for determining whether a microorganism produces carbapenem hydrolysing β-lactamase, comprising an assay disc impregnated with (7R)-7-[(2)-2-(2-aminothiazole-4- yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dinitrostyryl)-3-cephem-4-carboxylic acid or a salt thereof and an extended spectrum β-lactamase (ESBL) inhibitor, an AmpC inhibitor, or their mixture.

The present invention also relates to a microtiter plate containing a well or series of wells containing (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3 -(2,4-dinitrostyryl)-3-cephem-4-carboxylic acid or its salt, and its use in detecting the presence of carbapenemase producers in the test sample.

The present invention also relates to vials or microcentrifuge tubes containing (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2, 4-dinitrostyril)-3-cephem-4-carboxylic acid or its salt, and their use in detecting the presence of carbapenemase producers in the test sample.

Detailed description of the invention

Methods that allow rapid detection of carbapenemase-producing bacteria are necessary in order to take the necessary steps to prevent the spread of antibiotic resistance and to maintain the efficacy of antibiotics such as penems and carbapenems. The method of the present invention satisfies this need by providing the user with interpretable results to quickly and reliably determine whether a sample contains carbapenemase-producing bacteria.

In particular, the method may be suitable for providing a rapid and reliable phenotypic test to detect the presence of carbapenemase-producing Enterobacteriales, Pseudomonas and Acinetobacter spp. Accordingly, the present invention preferably relates to a method suitable for providing a rapid and reliable phenotypic test to detect the presence of carbapenemase-producing bacteria, such as Pseudomonas aeruginosa, which cannot be quickly and reliably tested using other known methods and/or tests.

Thus, according to a first aspect, the present invention relates to a method for detecting the presence of carbapenemase-producing Enterobacteriales, Pseudomonas, and Acinetobacter in a sample, comprising: providing a sample in which the presence of carbapenemase-producing Enterobacteriales, Pseudomonas, or Acinetobacter is suspected; 7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dinitrostyryl)-3-cephem-4-carboxylic acid or its salts and detecting a color change in the reaction medium when carbapenemase-producing Enterobacteriales, Pseudomonas, or Acinetobacter are present in the test sample.

The method according to the present invention can be characterized by 100% sensitivity and 100% specificity in relation to the detection of carbapenemase production in representatives of Enterobacteriales, Pseudomonas and Acinetobacter spp.

The method according to the present invention is based on the idea that (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4- dinitrostyril)-3-cephem-4-carboxylic acid or a salt thereof can be hydrolyzed by a β-lactamase such as carbapenemase. Hydrolysis of ([(7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dinitrostyryl)-3-cephem -4-carboxylic acid results in the observed color change from yellow to red.

Preferably, the method of the present invention is carried out on samples obtained from a subject suspected of being infected with carbapenemase-producing bacteria. The sample may be any biological sample obtained from the subject, such as fluid, tissue, or cell samples. Preferably, the sample may be from an isolated cell culture or is a urine sample. The sample can be obtained using known methods from the subject, which may be a mammal. Preferably, the subject from which the sample is obtained is a human.

The method of the present invention can be used to detect any carbapenemase-producing Enterobacteriales, Pseudomonas, or Acinetobacter bacteria. Preferably, the carbapenemase-producing bacteria are clinically important, such as bacteria that are responsible for nosocomial or community-acquired infections.

Typically, the concentration of (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dinitrostyryl)-3- cephem-4-carboxylic acid or its salt, the substrate used in the method according to the present invention, is from 0.1 mg/ml to 10 mg/ml, more preferably from 1 mg/ml to 5 mg/ml, and even more preferably, from 2 mg/ml to 3 mg/ml.

In some embodiments, carbapenemase-producing bacteria are lysed prior to reaction with (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-( 2,4-dinitrostyryl)-3-cephem-4-carboxylic acid or a salt thereof. The lysis of bacteria can be carried out using any known technique. Optionally, the number of Enterobacteriales, Pseudomonas or Acinetobacter spp. may be resuspended in Protein Extraction Buffer/Reagent in a microcentrifuge tube or vial prior to mixing. Mixing can be done by vortexing for a suitable period to ensure complete mixing. Preferably, mixing can be achieved by vortexing the sample for a period of about 2 to about 10 seconds, such as 5 seconds. The tube can then be incubated for a period of 5 to 30 minutes at a suitable temperature. Optionally, the tube may be incubated for a period of about 10 minutes. Suitable temperatures may be from about 25°C to about 45°C. Preferably, the tube may be incubated at about 35°C to about 37°C.

In some embodiments, sample lysis is facilitated with a combination of a polymyxin, glycopeptide, and polypeptide antibiotic.

Reaction of a sample, which may be a lysed sample, with (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2, 4-dinitrostyril)-3-cephem-4-carboxylic acid or a salt thereof can be carried out for a period of time sufficient to observe a color change. Preferably, the color change should be observed in less than 1 hour. More preferably, the color change can be observed in less than 30 minutes. Most preferably, the color change can be observed over a period of about 5 to about 20 minutes.

Sample reaction with (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dinitrostyryl)-3-cephem The -4-carboxylic acid or salt thereof can be carried out at any suitable temperature. Preferably, the reaction can be carried out at a temperature of from about 5°C to about 40°C. For example, the reaction can be carried out at a temperature of from about 15°C to about 30°C, such as at room temperature. Preferably, the reaction can be carried out at a temperature of from about 20°C to about 37°C and most preferably at 35°C.

Chromogenic cephalosporin, (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dinitrostyril)-3-cephem -4-carboxylic acid, or a salt thereof, is also sensitive to hydrolysis by extended spectrum beta-lactamases (ESBL) and AmpC. However, the effects of the ESBL and AmpC enzymes can be eliminated by the addition of suitable inhibitor compounds. Accordingly, when such inhibitors are present, a color change indicates the presence of carbapenemase.

Accordingly, in accordance with one embodiment of the method, an extended spectrum β-lactamase (ESBL) inhibitor, an AmpC inhibitor, or a mixture thereof is further provided. Any suitable inhibitor may be used. Some suitable inhibitors include clavulanic acid, cloxacillin, and mixtures thereof. Optionally, the inhibitor comprises at least one of clavulanic acid at a concentration of about 30 μg/ml to about 50 μg/ml and cloxacillin at a concentration of from about 300 μg/ml to about 500 μg/ml. In the case where the inhibitor contains clavulanic acid and cloxacillin, they can be mixed in equal parts to obtain a mixture of inhibitors. Alternatively, different amounts and/or additional components may be used. In addition, either carbapenem or penem can be used alone or in combination with both ESBL inhibitors and AmpC inhibitors to further eliminate ESBL and AmpC activity.

The choice of inhibitors that inhibit extended-spectrum beta-lactamase (ESBL) and AmpC allows visualization of the full spectrum of carbapenemase expression.

According to one embodiment of the present invention, (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dinitrostyril )-3-cephem-4-carboxylic acid or a salt thereof can be dissolved in a solution containing at least one of an organic solvent, an organic acid, and zinc sulfate. Optionally, the organic solvent may be a polar aprotic solvent. A suitable solvent may be DMSO. The organic acid may be any suitable organic acid. Suitable organic acids may include organic acids from the group of linear saturated dicarboxylic acids such as oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid or sebacic acid.

It will be understood that the sample reaction can be carried out using a sequential series of steps, for example, including at least some of preparing a mixture of inhibitors, dissolving (7R)-7-[(z)-2-(2-aminothiazol-4-yl )-(Z)-2(methoxyimino)acetamide]-3-(2,4-dinitrostyryl)-3-cephem-4-carboxylic acid or a salt thereof in a solution containing at least one of an organic solvent, an organic acid and a sulfate zinc, suspending the amount of carbapenemase-producing Enterobacteriales, Pseudomonas and Acinetobacter in the amount of protein extraction reagent for testing and/or heating the suspended carbapenemase-producing Enterobacteriales, Pseudomonas and Acinetobacter to obtain a live sample and adding to the sample the lysed material of the solution (7R)-7- [(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dinitrostyryl)-3-cephem-4-carboxylic acid (or its salts ) with the amount of a mixture of inhibitors.

According to other embodiments, the reaction process may be carried out by a reaction process other than sequential, wherein three or more of (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z) -2(methoxyimino)acetamide]-3-(2,4-dinitrostyryl)-3-cephem-4-carboxylic acid or its salt, organic solvent, organic acid, zinc sulfate is added to a sample of carbapenemase-producing Enterobacteriales, Pseudomonas and Acinetobacter and lyse in situ simultaneously with the reaction process.

For example, the reaction of a sample can be carried out using a "one-pot" synthesis technique, in which it is possible to react an unlysed sample with (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2 (methoxyimino)acetamide]-3-(2,4-dinitrostyryl)-3-cephem-4-carboxylic acid or a salt thereof, an organic solvent and an organic salt. Optionally, the organic solvent may be DMSO and the organic acid may be a linear saturated dicarboxylic acid such as succinic acid.

According to a second aspect, the present invention relates to the use of (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2.4 -dinitrostyril)-3-cephem-4-carboxylic acid or a salt thereof to detect the presence of carbapenemase-producing Enterobacteriales, Pseudomonas or Acinetobacter in the sample.

Optionally, (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dinitrostyryl)-3-cephem- The 4-carboxylic acid or salt thereof may be in powder form. ([(7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dinitrostyril)-3-cephem- The 4-carboxylic acid or salt thereof can be dissolved in a mixture of DMSO and succinic acid followed by the addition of zinc sulfate For example, (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)- 2(methoxyimino)acetamide]-3-(2,4-dinitrostyryl)-3-cephem-4-carboxylic acid or a salt thereof can be dissolved in 1 part DMSO with 3-20 parts of succinic acid followed by addition of zinc sulfate to give the final concentration 0.1 mM to 10 mM Preferably (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2, 4-dinitrostyryl)-3-cephem-4-carboxylic acid or a salt thereof can be dissolved in 1 part DMSO with 5-9 parts succinic acid Optionally, (7R)-7-[(z)-2-(2-aminothiazole- 4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dinitrostyryl)-3-cephem-4-carboxylic acid or a salt thereof can be dissolved in 1 part DMSO with 7 parts succinic acid followed by by adding zinc sulfate to give a final concentration of 1 mM .

The amount of lysed sample or whole cell sample can be added in approximately equal parts to a solution of (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3- (2,4-dinitrostyryl)-3-cephem-4-carboxylic acid or a salt thereof. A lysed or whole cell sample can be added to a solution of (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dinitrostyril )-3-cephem-4-carboxylic acid or a salt thereof at room temperature. Optionally, about 20 µl to about 100 µl of lysed sample and solution of (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]- may be used. 3-(2,4-dinitrostyryl)-3-cephem-4-carboxylic acid or a salt thereof.

You can also add the amount of inhibitor. Any suitable inhibitor, or mixtures thereof, may be used in amounts from 20 μl to about 200 μl. Preferably, about 50 µl to about 150 µl of inhibitor may be used. Optionally, about 100 µl of an inhibitor containing equal parts clavulanic acid and cloxacillin adjusted to a final concentration of about 40 µg/ml and 400 µg/ml can be added.

Alternatively, a different inhibitor or mixture of inhibitors or a mixture thereof in different ratios can be added at an equivalent final concentration.

The contents of the resulting solution can be monitored for a period of time up to an hour and preferably up to 30 minutes to determine if a color change occurs. Preferably, the solution may only need to be monitored for a period of 5 to 30 minutes.

The appearance of a yellow to orange/red color change may indicate the presence of carbapenemase-producing Enterobacteriales, Pseudomonas, or Acinetobacter in the test sample.

In order to investigate whether a carbapenemase-producing Enterobacteriales, Pseudomonas, or Acinetobacter is present in the test sample, the amount of Enterobacteriales, Pseudomonas, or Acinetobacter spp. can be suspended in protein extraction buffer/reagent. The solution can then be mixed and incubated to obtain a lysed sample. A portion of the lysed sample can be added to the amount of (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dinitrostyril) -3-cephem-4-carboxylic acid or a salt thereof, and optionally the amount of a mixture of inhibitors, a positive test being confirmed by observing a color change.

The applied amount of representatives of Enterobacteriales, Pseudomonas or Acinetobacter spp. may be from about 0.1 μl to about 10 μl. For example, about 0.2 µl, 0.5 µl, 1 µl, 2 µl, or 5 µl can be used. The amount of protein extraction reagent used can be from 50 to 250 µl. For example, about 50 µl, 75 µl, 100 µl, 150 µl can be used. Preferably, about 1 µl of an Enterobacteriales, Pseudomonas or Acinetobacter spp. representative may be used. and about 100 µl of protein extraction buffer/reagent. Any suitable protein extraction buffer/reagent may be used.

Mixing can be achieved using any suitable technique, including, without limitation, using mixing, shaking and mixing on a vortex mixer. For example, the solution can be vortexed for a period of about 1 to 60 seconds, such as for about 5 seconds. Mixing may occur before and/or during incubation to obtain a lysed sample. Incubation can be carried out at any suitable temperature and for any suitable period of time. For example, incubation can be carried out at a temperature of about 30 to 45°C, preferably about 35 to 37°C, for about 1 to about 30 minutes, preferably for about 10 minutes.

Optionally, the test can be performed on a non-lysed sample.

Any suitable portion of the lysed (or non-lysed) sample can be added to any suitable amount of (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]- solution 3-(2,4-dinitrostyryl)-3-cephem-4-carboxylic acid or a salt thereof, optionally with an amount of a mixture of inhibitors. Optionally, from about 10 µl to about 100 µl, for example, about 50 µl, of a lysed (or non-lysed) sample can be added to a solution of (7R)-7-[(z)-2-(2-aminothiazol-4-yl)- (Z)-2(methoxyimino)acetamide]-3-(2,4-dinitrostyryl)-3-cephem-4-carboxylic acid or a salt thereof in an amount of from about 20 µl to about 100 µl, for example, about 50 µl, optional , in addition to about 20-200 µl, eg about 100 µl, of the mixture of inhibitors.

Preferably, sample, solution (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dinitrostyryl)-3 α-cephem-4-carboxylic acid or a salt thereof and optionally a mixture of inhibitors can be added at room temperature.

According to a third aspect, the present invention relates to a kit for determining whether a microorganism produces carbapenem-hydrolysing β-lactamase, which may comprise an assay disc impregnated with ([(7R)-7-[(z)-2-(2-aminothiazole -4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dinitrostyryl)-3-cephem-4-carboxylic acid) or a salt thereof and an extended spectrum β-lactamase inhibitor (ESBL), an AmpC inhibitor; or a mixture thereof.

According to a further aspect, the present invention relates to a microfluidic device or an electrochemical sensor device for detecting the presence of carbapenemase-producing Enterobacteriales, Pseudomonas, or Acinetobacter, the device comprising a substrate that is (7R)-7-[(z)-2-( 2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dinitrostyryl)-3-cephem-4-carboxylic acid or a salt thereof.

The microfluidic device may be any device that produces results that can be read as a phenotypic characteristic. For example, the microfluidic device may be a microcapillary film.

The electrochemical sensor device can be any device that will signal upon hydrolysis of (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3 -(2,4-dinitrostyryl)-3-cephem-4-carboxylic acid or a salt thereof. For example, an electrochemical sensor device may be an electrode, a chip, a kit, or an ion-charged membrane.

The reaction of the lysed sample with a set of reagents is carried out for a period of time sufficient to observe a color change in a solution of (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino) acetamide]-3-(2,4-dinitrostyryl)-3-cephem-4-carboxylic acid or a salt thereof, when carbapenemase-producing Enterobacteriales, Pseudomonas or Acinetobacter are present in the test sample. Typically, a color change is observed in less than 1 hour and more preferably in less than 30 minutes from the moment the sample is mixed with the buffer/reagent. As a rule, the reaction of the sample with a set of reagents is carried out at a temperature including from 5°C to 40°C, preferably from 15°C to 30°C, and more preferably at room temperature.

The reagent kit can be used to provide a very fast way to identify infection by carbapenemase-producing Enterobacteriaceae, Pseudomonas, or Acinetobacter bacteria with high certainty and accuracy in accordance with the method of the present invention.

In FIG. 1 shows the results of a phenotypic study on Pseudomonas spp.

In FIG. 2 shows the results of a phenotypic study of Acinetobacter baumanii.

In FIG. 3 shows a comparative study of Pseudomonas and Acinetobacter spp. using the method according to the present invention and standard methods.

In FIG. 4 shows the results of a phenotypic study to identify carbapenemase-producing Enterobacteriales.

The present invention will be further illustrated with reference to the following examples.

EXAMPLES

Example 1

Test solution containing a mixture of inhibitors and ([(7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4- dinitrostyril)-3-cephem-4-carboxylic acid was prepared and used to determine the presence of carbapenemase-producing Pseudomonas or Acinetobacter in a number of test samples, as set out in Tables 1 and 2.

The test solution was composed as follows:

1) clavulanic acid and cloxacillin were at a final concentration of 40 μg/ml and 400 μg/ml and mixed together in equal parts to obtain a mixture of inhibitors;

2) (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dinitrostyril)-3-cephem- The 4-carboxylic acid or its salt in powder form was dissolved in 1 part DMSO with 7 parts dicarboxylic acid followed by addition of zinc sulfate to a final concentration of 1 mM;

3) 1 µl of samples of either Pseudomonas or Acinetobacter spp. the seed loop was suspended in 100 µl of commercially available protein extraction buffer/reagent or 100 µl of extraction buffer containing 50 mM Tris HCl, 100 mM NaCl and 1% Triton X100 in a microcentrifuge tube;

4) suspended representatives of Pseudomonas or Acinetobacter spp. mixed by vortexing for a period of 5 seconds before heating the sample in a microcentrifuge to 35°C-37°C and incubating for a period of 10 minutes to obtain a lysed sample;

5) 50 µl of the lysed sample was added to 50 µl of a solution of (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2, 4-dinitrostyril)-3-cephem-4-carboxylic acid or a salt thereof and 100 μl of a mixture of inhibitors in a well of a 96-well microtiter plate at room temperature;

6) the contents of the well were mixed by pipetting while adding the cell lysate;

7) the sample was monitored visually for a period of up to 30 minutes, fixing the color of the solution after 5, 10, 20 and 30 minutes from the moment the sample was mixed with the solution ([(7R)-7-[(z)-2-(2-aminothiazole- 4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dinitrostyryl)-3-cephem-4-carboxylic acid or a salt thereof.

As shown in Table 1, (7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dinitrostyril)- 3-cephem-4-carboxylic acid or its salt successfully detected all 9 carbapenemase-producing Pseudomonas spp. The sensitivity of the method according to the present invention was 100%, as was the specificity.

Corresponding phenotypic results for Pseudomonas spp. shown in Fig. one.

These tests show that the method according to the present invention is very sensitive in terms of detection of NDM-1, IMP and VIM in Pseudomonas spp. Moreover, it has been shown that the yellow color of a solution of (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4- dinitrostyryl)-3-cephem-4-carboxylic acid or its salt undergoes a significant red color change within 5 minutes after addition of samples of lysed bacteria at room temperature. Although a more intense red color may appear over longer periods of time, a color change after 5 minutes is sufficient to show a positive reaction.

As shown in Table 2, (7R)-7-[(Z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dinitrostyryl)- 3-cephem-4-carboxylic acid or its salt successfully detected all 5 carbapenemase-producing Acinetobacter baumanii. The sensitivity of the method according to the present invention was 100%, as was the specificity.

The corresponding phenotypic results for Acinetobacter baumanii are shown in FIG. 2.

These tests show that the method according to the present invention is very sensitive in terms of detection of OXA in Acinetobacter spp. Moreover, it has been shown that the yellow color of a solution of (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4- dinitrostyryl)-3-cephem-4-carboxylic acid or its salt undergoes a significant red color change within 5 minutes after addition of samples of lysed bacteria at room temperature. Although a more intense red color may appear over longer periods of time, a color change after 5 minutes is sufficient to show a positive reaction.

Example 2

The test solution was formulated according to the method of Example 1 and used to test five Pseudomonas isolates; of which 4 isolates were resistant to carbapenems and 1 was a non-carbapenemase producing organism. Of the 4 carbapenemase-producing Pseudomonas isolates, 2 were NDM-1 while the remaining 2 were VIM. Six Acinetobacter isolates were also studied, of which 5 isolates were carbapenem resistant and 1 was a non-carbapenemase producing organism. All 5 carbapenemase-producing Acinetobacter isolates were OXA.

Appropriate tests for each isolate of Pseudomonas and Acinetobacter were also performed using a commercially available colorimetric test for the detection of strains with reduced sensitivity to carbapenems, known to the skilled person in the art. Following the standard procedure, comparative studies based on the colorimetric technique were carried out at 35° C.-37° C., while tests carried out in accordance with the method according to the present invention were carried out at room temperature.

The results of these tests are presented in Table 3, with the observed color changes being shown in FIG. 3.

The test included a negative control isolate of Pseudomonas and Acinetobacter, consisting of a carbapenemase-negative isolate of Pseudomonas aeruginosa and Acinetobacter iwoffi. All other isolates were carbapenemase producers.

As shown in Table 3, of the 11 organisms tested in the comparison, (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3- (2,4-dinitrostyryl)-3-cephem-4-carboxylic acid or a salt thereof and a commercially available pH dependent diagnostic method, (7R)-7-[(z)-2-(2-aminothiazol-4-yl) -(Z)-2(methoxyimino)acetamide]-3-(2,4-dinitrostyryl)-3-cephem-4-carboxylic acid or its salt successfully detected all nine of the Pseudomonas and Acinetobacter carbapenemase-producing isolates, while tests no carbapenemase production was detected in a single Acinetobacter isolate (highlighted in the table) using standard procedures. The false negative that was incorrectly identified using the standard test was OXA-27.

Accordingly, it is shown that the standard method has a specificity of 100% but only 89% sensitivity, while the method according to the present invention has a sensitivity and specificity of 100%. Thus, the method of the present invention provides a rapid method for detecting the presence of carbapenemase-producing Pseudomonas or Acinetobacter in a test sample with improved sensitivity compared to known techniques.

Example 3

The two test solutions were formulated according to the method of Example 1. One of the solutions was exactly as described in Example 1 and the other solution used the chromogenic cephalosporin HMRZ-86 instead of (7R)-7-[(z)-2-( 2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dinitrostyryl)-3-cephem-4-carboxylic acid or a salt thereof.

Both test solutions were then used to test 68 Pseudomonas isolates; of which 8 isolates were resistant to carbapenems and 60 were not producers of carbapenemase. Of the 8 carbapenemase-producing Pseudomonas isolates, 4 were NDM-1, 2 were VIM, while the remaining 2 were IMP.

Study with both solutions using either HMRZ-86 or (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3- (2,4-dinitrostyryl)-3-cephem-4-carboxylic acid was carried out in accordance with the method according to the present invention (carried out at room temperature). The test results are presented in Table 4.

As shown in Table 4, of the 8 carbapenemase-producing organisms from the genus Pseudomonas tested in the comparison, (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino) acetamide]-3-(2,4-dinitrostyryl)-3-cephem-4-carboxylic acid with HMRZ, (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z) -2(methoxyimino)acetamide]-3-(2,4-dinitrostyryl)-3-cephem-4-carboxylic acid successfully detected all eight of the carbapenemase-producing Pseudomonas isolates, while tests using HMRZ-86 instead of (7R) -7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dinitrostyryl)-3-cephem-4-carboxylic acid is not two NDM-1 producing isolates were identified (highlighted in Table 4).

Accordingly, the method of the present invention was shown to have a sensitivity and specificity of 100%, while the candidate chromogenic substrate HMRZ-86 resulted in 75% sensitivity. Thus, the method according to the present invention using (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4- dinitrostyril)-3-cephem-4-carboxylic acid is more sensitive in the case of rapid detection of carbapenemase-producing Pseudomonas or Acinetobacter in the studied samples.

Example 4

Dicarboxylic acid buffer stabilized test solution containing ([(7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2, 4-Dinitrostyril)-3-cephem-4-carboxylic acid, a mixture of inhibitors, zinc sulfate, and excipient were prepared and used to determine the presence of carbapenemase-producing Enterobacteriales, Pseudomonas, or Acinetobacter in the 147 test isolates as set out in Table 5.

The test solution was composed as follows:

1) (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dinitrostyril)-3-cephem- The 4-carboxylic acid in powder form was dissolved in 1 part DMSO with 7 parts dicarboxylic acid;

2) the solution from step 1 above was then stabilized with a buffer by further addition of dicarboxylic acid in a ratio of 1 part test solution to 4 parts dicarboxylic acid;

3) clavulanic acid and cloxacillin were at a final concentration of 40 μg/ml and 400 μg/ml and mixed together in equal parts to obtain a mixture of inhibitors;

4) zinc sulfate and PEG 8000 were added to the mixture of inhibitors to final concentrations of 1 mm and 0.5%, respectively;

5) a mixture containing 2 mm polymyxin, 4 mm glycopeptide and 1.5 mm polypeptide antibiotics was also dissolved in a solution containing a mixture of inhibitors;

6) Volumes of 2.8 ml of a solution containing the mixture of inhibitors, zinc sulfate, PEG 8000 and the mixture of antibiotics described in step 5 above were then lyophilized as pellets for 72 hours.

The presence of carbapenemase was then determined as follows:

(a) recover the precipitate by adding 3.5 ml of the test solution from step 2 above;

(b) ensure complete dissolution of the precipitate at room temperature within 1 minute and mix the contents by light agitation on a vortex mixer for 10 seconds;

(c) the reconstituted solution must be yellow; if the solution is any other color, do not use it;

(d) dispense 500 µl of the reconstituted solution into microcentrifuge tubes; One tube per analysis.

(e) using a clean fresh culture of the test organism, add 1 µl of the organism sample from the seeding loop to the microcentrifuge tube and mix well with a vortex mixer for 10 seconds;

(f) incubate at 35-37°C for 10 minutes and record the color of the test solution.

110 representatives of Enterobacteriales were studied; of which 72 isolates were carbapenemase producers, and 38 were not carbapenemase producers. Of the isolates studied, 57 were Klebsiella spp. [12 ESBL, 5 AmpC, 10 OXA, 13 KPC and 17 MβL (6 NDM, 6 IMP and 5 VIM)], 24 were Escherichia coli [8 ESBL, 5 AmpC, 5 OXA, 1 KPC and 5 MβL(2 VIM) , 2 NDM and 1 IMP)], 23 were Enterobacter spp. [6 ESBL, 10 AmpC, 2 OXA, 4 MβL (2 VIM, 2 NDM and 1 KPC)], 3 - Citrobacter spp. [all NDM], 1 - Kluvyera spp. [NDM producer] and tested 2 isolates of Salmonella spp.; both of which were producers of AmpC [enzymes other than carbapenemase].

- 23 isolates of Pseudomonas spp. were studied; of which 8 isolates were producers of MβL carbapenemase containing 2 NDM, 2 IMP and 4 VIM. Fifteen Pseudomonas isolates did not produce carbapenemase [14 of which had no enzyme-mediated mechanism, while one isolate produced AmpC].

- 14 isolates of Acinetobacter spp. were studied; of which 13 isolates were carbapenemase producers, and one was not a carbapenemase producer. Among carbapenemase producers, there were 12 oxacillinase-producing isolates [4 OXA-23, 1 OXA-25, 1 OXA-26, 1 OXA-27, 1 OXA-58 and co-producers such as 2 OXA23 + OXA-51, 1 OXA-23 + OXA-27 + OXA-51 and 1 OXA-95 + AmpC]. One of the carbapenemase-producing Acinetobacter isolates was a MβL producer.

As shown in Table 5, out of a total of 147 isolates tested, the method of the present invention detected 81 of 83 carbapenemase-producing Enterobacteriales, Pseudomonas and Acinetobacter isolates. This correlates with an overall sensitivity of 97.59% and 100% specificity for a broad spectrum of Gram-negative bacteria. At the same time, a commercially available kit was considered unsuitable for use with Pseudomonas aeruginosa (Henrichs et at, 2015), while another kit, the main competitor, which suggests the use of chromogenic HMRZ-86, showed only 64.9% sensitivity and 90% specificity. regarding the detection of carbapenemase-producing Enterobacteriaceae (Mancini et at, 2017). Therefore, at present, the present invention is the only method for the rapid colorimetric detection of carbapenemase with very high sensitivity and specificity, which is suitable not only for use with isolates of Pseudomonas and Acinetobacter spp., but also extends to Enterobacteriales.

Claims (16)

1. A method for detecting the presence of carbapenemase-producing Enterobacteriales, Pseudomonas and Acinetobacter in a biological sample, comprising: providing a sample suspected of having carbapenemase-producing Enterobacteriales, Pseudomonas, or Acinetobacter, reacting said sample with a solution of (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino )acetamide]-3-(2,4-dinitrostyryl)-3-cephem-4-carboxylic acid) or a salt thereof, wherein the reaction is carried out in the presence of an extended spectrum β-lactamase inhibitor (ESBL), an AmpC inhibitor, or a mixture thereof, and detection color changes from yellow to orange/red in the reaction medium when carbapenemase-producing Enterobacteriales, Pseudomonas or Acinetobacter are present in the test sample, and the color change occurs within 1 hour of sample reaction. 2. The method according to claim 1, wherein the biological sample is selected from the group consisting of a biological sample such as urine, or, as a rule, from an isolated cell culture. 3. The method according to any one of the preceding claims, the method further comprising the step of lysing the sample before or simultaneously with the reaction with (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2( methoxyimino)acetamide]-3-(2,4-dinitrostyril)-3-cephem-4-carboxylic acid) or a salt thereof. 4. The method according to paragraphs. 1, 2, and the lysis of the sample occurs in the presence of (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4 -dinitrostyril)-3-cephem-4-carboxylic acid) or a salt thereof. 5. The method of claim 4, wherein sample lysis is facilitated by a combination of a polymyxin, glycopeptide, and polypeptide antibiotic. 6. The method according to any of the preceding claims, wherein the reaction of the sample with ([(7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3- (2,4-dinitrostyryl)-3-cephem-4-carboxylic acid) or a salt thereof is carried out for a period of time sufficient to observe a color change, said period of time being less than 30 minutes and preferably from about 5 to about 20 minutes. 7. The method according to any preceding claim, wherein the reaction of the sample with ([(7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3- (2,4-dinitrostyryl)-3-cephem-4-carboxylic acid) or a salt thereof is carried out at a temperature of from about 5°C to about 40°C, more preferably from about 20°C to about 37°C, and most preferably at 35°C. 8. The method according to any preceding paragraph, wherein ([(7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2, 4-dinitrostyril)-3-cephem-4-carboxylic acid) or a salt thereof is used in combination with carbapenem or penem alone or in combination with an extended spectrum β-lactamase inhibitor (ESBL), an AmpC inhibitor, or a mixture thereof. 9. The method of any one of the preceding claims, wherein the extended spectrum inhibitor is clavulanic acid and the AmpC inhibitor is cloxacillin or a mixture thereof. 10. The method of any preceding claim, wherein the inhibitor comprises at least one of clavulanic acid at a concentration of about 30 μg/ml to about 50 μg/ml and cloxacillin at a concentration of from about 300 μg/ml to about 500 μg/ml. 11. The method according to any preceding paragraph, wherein ([(7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2, 4-dinitrostyryl)-3-cephem-4-carboxylic acid) or a salt thereof is dissolved in a solution containing at least one of an organic solvent, an organic acid, and zinc sulfate. 12. The method of claim 8, wherein the organic solvent is a polar aprotic solvent, preferably DMSO, and wherein the organic acid is a linear saturated dicarboxylic acid. 13. Application of ([(7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dinitrostyril)-3 -cephem-4-carboxylic acid) or salt thereof to detect the presence of carbapenemase-producing Enterobacteriales, Pseudomonas and Acinetobacter in the sample. 14. Application of ([(7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dinitrostyril)-3 -cephem-4-carboxylic acid) or a salt thereof according to claim 13, wherein ([(7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino) acetamide]-3-(2,4-dinitrostyryl)-3-cephem-4-carboxylic acid) or a salt thereof are used in combination with carbapenem or penem alone or in combination with an extended spectrum β-lactamase inhibitor (ESBL), AmpC or their mixture. 15. A kit for determining whether a microorganism produces carbapenem hydrolyzing β-lactamase containing a vial that contains ([(7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)- 2(methoxyimino)acetamide]-3-(2,4-dinitrostyryl)-3-cephem-4-carboxylic acid) or salt thereof, a microtiter plate having wells that are coated with ([(7R)-7-[(z) -2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dinitrostyryl)-3-cephem-4-carboxylic acid) or its salt, or disk for assay impregnated with HMRZ-98 ([(7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dinitrostyril )-3-cephem-4-carboxylic acid) or a salt thereof, wherein the vial, microtiter plate and/or assay disk contains an extended spectrum β-lactamase inhibitor (ESBL), an AmpC inhibitor, or a mixture thereof.

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