DETECTION OF CARBAPENEMASE-PRODUCING

ENTEROBACTERIALES, PSEUDOMONAS AND ACINETOBACTER SPECIES USING A CHROMOGEN

FIELD OF INVENTION

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

BACKGROUND 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 carbapenemases confer resistance in clinically important bacteria to carbapenems or decreased susceptibility to carbapenems. Bacteria that produce carbapenemase are therefore of interest clinically and methods for their early detection are required in order to prevent their spread and to reduce multidrug and pandrug resistance.

Two tests are widely used clinically to detect carbapenemase producing bacteria; the "Etest®" and other MIC strip products and the "Modified Hodge-Test". In the "Etest®", indicator carbapenem and carbapenem plus enzyme inhibitor antimicrobial agents against a test microorganism are provided in opposing predefined gradients on agar and used to determine the Minimum Inhibitory Concentration (MIC) of the antimicrobial agent and the inhibitory concentration ratio. In the "Modified Hodge-Test" carbapenemase producing bacteria are detected when reporter bacteria grow towards a carbapenem containing disk to produce a characteristic "clover-leaf growth pattern around the disk. This phenomenon can be mediated by the carbapenem inactivating enzymes produced by the test isolate. Molecular detection techniques for carbapenemase genes have also been used. However, all three tests have their drawbacks. The "Etest®" and the "Modified Hodge-Test" are neither sensitive nor specific enough whilst molecular detection techniques are complicated and expensive. Moreover, all three detection methods are highly time consuming with up to 24 hours being the typical timescale for determining whether a sample contains carbapenemase producing bacteria. This is unsatisfactory when controlling nosocomial acquired infections and the transfer of drug resistance.

Recently acido-colorimetric techniques have been developed for the detection of carbapenemase producing bacteria which are reported as significantly lowering the assay time compared to "Etest®", "Modified Hodge-test" and molecular detection techniques. WO2012/175637 describes such a method in which specifically, assay times of less than 2 hours are reported. However, current rapid phenotypic tests show poor sensitivity and specificity for the detection of carbapenemase- producing Pseudomonas (Heinrichs et al., 2015) and Acinetobacter in a sample. In particular, false negatives may be obtained.

Also, rapidly detecting carbapenemase producing Enterobacteriaceae is key to limiting the spread of these organisms and although there are both phenotypic and molecular methods available for detecting these, no single detection method has proven ideal for ai l situations (Lutgring et Limbago, 2016). According to the European Committee on Antimicrobial Susceptibility Testing (EUCAST, 2018), recent taxonomic studies have narrowed the definition of the family Enterobacteriaceae. Consequently, the Enterobacteriales are an order of gram-negative bacteria that includes only one family; Enterobacteriaceae. Therefore, the present invention will make reference to the order Enterobacteriales.

Consequently, there is a need to provide a method for detecting the presence of carbapenemase- producing Enterobacteriales, Pseudomonas and Acinetobacter in a sample which is not only highly specific and sensitive, but may also be easily utilized clinically.

SUMMARY OF THE INVENTION

The inventors have found that by conducting a phenotypic test using the chromogenic cephalosporin (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)aceta mide]-3- (2,4-dinitro styryl)-3-cephem-4 carboxylic acid or a salt thereof, it is possible to detect the presence of carbapenemase producing Enterobacteriales, Pseudomonas and Acinetobacter in very short timescales. In particular, the detection process found by the inventors does not require pre-exposure of isolates to any agents that might induce or inhibit expression prior to testing for the presence of carbapenemase, and which thus requires an additional information period.

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

Thus, in a first aspect the present invention provides a method for detecting the presence of carbapenemase-producing Enterobacteriales, Pseudomonas and Acinetobacter in a sample from a patient, comprising: providing a sample suspected of having carbapenemase-producing Enterobacteriales, Pseudomonas or Acinetobacter, reacting the 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, and detecting a color change in the reaction medium, wherein a change in color 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 provides the use of (7R)-7-[(z)-2-(2- aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dini trostyryl)-3-cephem-4 carboxylic acid or a salt thereof, for detecting the presence of carbapenemase-producing Enterobacteriales, Pseudomonas and Acinetobacter in a sample.

According to a third aspect the present invention also provides a kit for determining whether a microorganism produces a carbapenem-hydrolysing β-lactamase, comprising an assay disk impregnated with (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)- 2(methoxyimino)acetamide]-3-(2,4-dinitro styryl)-3-cephem-4 carboxylic acid or a salt thereof, and an extended spectrum β-lactam (ESBL) inhibitor, a AmpC inhibitor or a mixture thereof. The invention also relates to a microtitre plate comprising a well or a series of wells comprising (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)aceta mide]-3-(2,4- dinitro styryl)-3-cephem-4 carboxylic acid or a salt thereof and its use in detecting the presence of carbapenemase producers in a test sample.

Also, the invention also relates to vials or microfuge tubes comprising (7R)-7-[(z)-2-(2- aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dini tro styryl)-3-cephem-4 carboxylic acid or a salt thereof and its use in detecting the presence of carbapenemase producers in a test sample.

DETAILED DESCRIPTION OF THE INVENTION

Methods which allow rapid detection of carbapenemase-producing bacteria are needed in order to take necessary action to prevent the spread of antibiotic resistance and to preserve the efficacy of antibiotics such as penems and carbapenems. The method of the present invention addresses this need by providing a user with interpretable results to determine whether a sample has carbapenemase producing bacteria in a fast and reliable manner.

In particular, the method may be suitable for providing a rapid and reliable phenotypic test for detecting the presence of carbapenemase-producing Enterobacteriales, Pseudomonas and Acinetobacter spp. Accordingly, the present invention advantageously provides a method suitable for providing a rapid and reliable phenotypic test for detecting the presence of carbapenemase-producing bacteria, such as Pseudomonas aeruginosa which may not be rapidly and reliably tested for using other known methods and/or tests.

In a first aspect, the present invention therefore provides a method for detecting the presence of carbapenemase-producing Enterobacteriales, Pseudomonas and Acinetobacter in a sample, comprising: providing a sample suspected of having carbapenemase-producing Enterobacteriales, Pseudomonas or Acinetobacter, reacting the sample with a solution of (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)- 2(methoxyimino)acetamide]-3-(2,4-dinitro styryl)-3-cephem-4 carboxylic acid or a salt thereof, and detecting a color change in the reaction medium when carbapenemase- producing Enterobacterial es, Pseudomonas or Acinetobacter are present in the test sample.

The method of the present invention may have 100% sensitivity and 100% specificity for detecting carbapenemase production in Enterobacteriales, Pseudomonas and Acinetobacter spp.

The method of the invention is based on the concept that (7R)-7-[(z)-2-(2-aminothiazol-4- yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dinitrostyryl)-3-ce phem-4 carboxylic acid or a salt thereof may be hydrolysed by a β-lactamse such as a carbapenemase. The hydrolysis of ([(7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)ace tamide]-3-(2,4- dinitrostyryl)-3-cephem-4 carboxylic acid, results in an observable colour change from yellow to red.

Preferably, the method of the present invention is conducted on samples obtained from a subject which is suspected of being infected with carbapenemase producing bacteria. The sample may be any biological sample obtained from a subject such as fluids, tissues or cell samples. Preferably the sample may be from isolated cell culture or is a urine sample. The sample may be obtained by 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 may be used to detect any Enterobacteriales^ewifoworaxs' or Acinetobacter carbapenemase-producing bacteria. Preferably, the carbapenemase-producing bacteria are of clinical importance 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 a salt thereof, substrate used in the method of the 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. According to some embodiments, the carbapenemase-producing bacteria are lysed prior to reaction with (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)aceta mide]-3- (2,4-dinitrostyryl)-3-cephem-4 carboxylic acid or a salt thereof. Lysis of the bacteria may be performed by any known technique. Optionally, an amount of Enterobacterial es, Pseudomonas or Acinetobacter spp, may be resuspended in a protein extraction buffer/ reagent in a microfuge tube or vial prior to mixing. Mixing may be performed by vortexing for a suitable period to ensure thorough mixing. Preferably, mixing may be achieved by vortexing the sample for a period from about 2 to about 10 seconds, e.g. for 5 seconds. The tube may then be incubated for a period of between 5 and 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 from about 35 °C to about 37 °C.

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

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

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 may be carried out at any suitable temperature. Preferably, the reaction may be carried out from about 5 °C to about 40 °C. For example, the reaction may be carried out from about 15 °C to about 30 °C, such as at room temperature. Preferably, the reaction may be carried out from about 20 °C to about 37 °C, and most preferably at 35 °C.

The 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 is also susceptible to hydrolysis by extended spectrum beta-lactamases (ESBLs) and AmpCs. However, the effect of ESBL and AmpC enzymes may be eliminated by the addition of suitable inhibitor compounds. Accordingly, when such inhibitors are present, a change in colour indicates the presence of a carbapenemase.

Accordingly, in one embodiment the method further comprises an extended spectrum β- lactam (ESBL) inhibitor, an AmpC inhibitor or a mixture thereof. 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 between about 30 μg/ml to about 50 μg/ml and cloxacillin at a concentration of between about 300 μg/ml to about 500 μg/ml. Where in inhibitor comprises clavulanic acid and cloxacillin, these may be mixed together in equal parts to yield the inhibitor mix. Alternatively, different amounts and/or additional components may be used. Also, any carbapenem or penem could be used alone or in combinations, with both ESBL and AmpC inhibitors to further eliminate ESBL and AmpC activity.

The selection of inhibitors that inhibit extended spectrum beta-lactamases (ESBLs) and AmpCs allows for the visualisation of the whole spectrum of carbapenemase expression.

In one embodiment of the present invention, the (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 may be dissolved in a solution comprising at least one of an organic solvent, an organic acid and zinc sulphate. 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 those from the linear saturated dicarboxylic acid groupsuch as oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid, suberic acid, azelaic acid or sebacic acid.

It shall be understood that the reaction of the sample may be carried out utilizing a sequential sequence steps for example, including at least some of mixing an inhibitor mix, dissolving (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)aceta mide]-3-(2,4- dinitrostyryl)-3-cephem-4 carboxylic acid or a salt thereof in a solution comprising at least one of an organic solvent, an organic acid and zinc sulphate, suspending an amount of carbapenemase-producing Enterobacterial es, Pseudomonas and Acinetobacter in an amount of protein extractor reagent, for texting and or heating the suspended carbapenemase-producing Enterobacterial es, Pseudomonas and Acinetobacter to produce a live sample, and adding the lysed to sample to the (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) solution with an amount of the inhibitor mix.

In other embodiments, the reaction process may be carried out via a non-sequential reaction process, where three or more or 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, an organic solvent, an organic acid, zinc sulphate are added to a sample of carbapenemase-producing Enterobacterial es, Pseudomonas and Acinetobacter and lysed in situ simultaneously with the reaction process.

For example, the reaction of the sample may be carried out utilizing a "one-pot" synthesis technique in which an un-lysed sample may be reacted with (7R)-7-[(z)-2-(2- aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dini trostyryl)-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.

In a second aspect, the present invention provides the use of (7R)-7-[(z)-2-(2- aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dini trostyryl)-3-cephem-4 carboxylic acid or a salt thereof for detecting the presence of carbapenemase-producing Enterobacteriales, Pseudomonas or Acinetobacter in a sample.

The (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)aceta mide]-3-(2,4- dinitrostyryl)-3-cephem-4 carboxylic acid or a salt thereof may optionally be in powder form. The ([(7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)ace tamide]-3-(2,4- dinitrostyryl)-3-cephem-4 carboxylic acid or a salt thereof may be dissolved in a mixture of DMSO to and succinic acid, followed by the addition of zinc sulphate. For Example, the (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)aceta mide]-3-(2,4- dinitrostyryl)-3-cephem-4 carboxylic acid or a salt thereof may be dissolved in 1 part DMSO to from 3 to 20 parts succinic acid, followed by the addition of zinc sulphate to give a final concentration of from O. lmM to lOmM. Preferably, the (7R)-7-[(z)-2-(2- aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dini trostyryl)-3-cephem-4 carboxylic acid or a salt thereof may be dissolved in of 1 part DMSO to 5 to 9 parts succinic acid. Optionally, the (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 may be dissolved in a 1 part DMSO to 7 parts succinic acid, followed by the addition of zinc sulphate to give a final concentration of lmM.

An amount of lysed or whole cell sample may be added in approximately equal parts to the (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)aceta mide]-3-(2,4- dinitrostyryl)-3-cephem-4 carboxylic acid or a salt thereof solution. The lysed or whole cell sample may be added to the (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 solution at room temperature. Optionally, about 20μ1 to about ΙΟΟμΙ of the lysed sample and (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)aceta mide]-3-(2,4- dinitrostyryl)-3-cephem-4 carboxylic acid or a salt thereof solution may be used.

An amount of inhibitor may also be added. Any suitable inhibitor or mixtures thereof may be used in amounts from 20μ1 to about 200μ1. Preferably, about 50μ1 to about 150μ1 of inhibitor may be used. Optionally about ΙΟΟμΙ of an inhibitor comprising clavulanic acid and cloxacillin in equal parts made up to a final concentration of about 4C^g/ml and 400 μg/ml may be added.

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

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

The occurrence of a colour change from yellow to orange/red may indicate the presence of carbapenemase-producing Enterobacterial es, Pseudomonas or Acinetobacter in the test sample. In order to test whether a carbapenemase-producing Enterobacteriales, Pseudomonas or Acinetobacter is present in a test sample, an amount of Enterobacteriales, Pseudomonas or Acinetobacter spp may be suspended in a protein extraction buffer/reagent. The solution may then be mixed and incubated to produce a lysed sample. A portion of the lysed sample may be added to an amount 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 and optionally an amount of the inhibitor mix, with a positive test verified by the observance of a colour change.

The amount of Enterobacteriales, Pseudomonas or Acinetobacter spp used may be from about Ο. ΐ μΐ to about ΙΟμΙ. For example, about 0.2μ1, 0.5μ1, Ι μΐ, 2μ1 or 5μ1 may be used. The amount of protein extraction reagent used may be between 50 and 250μ1. For example, about 50μ1, 75μ1, ΙΟΟμΙ, 150μ1 may be used. Preferably about Ι μΐ of Enterobacteriales, Pseudomonas or Acinetobacter spp and about ΙΟΟμΙ of protein extraction buffer/reagent may be used. Any suitable protein extraction buffer/reagent may be used.

Mixing may be achieved using any suitable technique, including, but not limited to stirring, shaking and vortex mixing. For example, the solution may be vortex mixed for a period from about 1 to 60 seconds, such as for about 5 seconds. Mixing may occur prior to, and/or during, incubation to produce a lysed sample. Incubation may be performed at any suitable temperature and for any suitable length of time. For example, incubation may be performed at a temperature from about 30 to 45 °C, preferably about 35 to 37 °C, for about 1 to about 30 minutes, preferably about 10 minutes.

Optionally, the test may be performed on an un-lysed sample.

Any suitable portion of the lysed (or un-lysed) sample may be added to any suitable amount of the (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)aceta mide]-3- (2,4-dinitrostyryl)-3-cephem-4 carboxylic acid or a salt thereof solution, optionally with an amount of the inhibitor mix. Optionally, about ΙΟμΙ to about ΙΟΟμΙ, for example about 50μ1, of a lysed (or un-lysed) sample may be added to about 20μ to about ΙΟΟμΙ, for example about 50μ1 of the (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 solution, optionally in addition to about 20 to 200μ1, for example about ΙΟΟμΙ, of the inhibitor mix.

Preferably, the sample, (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 solution and optionally, inhibitor mix, may be added at room temperature.

In a third aspect, the present invention provides a kit for determining whether a microorganism produces a carbapenem-hydrolysing β-lactamase, may comprise an assay disk impregnated with ([(7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-

2(methoxyimino)acetamide]-3-(2,4-dinitro styryl)-3-cephem-4 carboxylic acid) or a salt thereof, and an extended spectrum β-lactam (ESBL) inhibitor, a AmpC inhibitor or a mixture thereof.

In a further aspect the present invention provides a microfluidic device or electrochemical sensing device for detecting the presence of a carbapenemase producing Enterobacteriales, Pseudomonas or Acinetobacter wherein the device comprises a substrate which is (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)aceta mide]- 3-(2,4-dinitro styryl)-3-cephem-4 carboxylic acid or a salt thereof.

The microfluidic device may be any device which gives results that may be read phenotypically. For example, the microfluidic device could be a micro-capillary film.

The electrochemical sensing device could be any device that will give a signal following the hyrolysis of (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)aceta mide]-3- (2,4-dinitro styryl)-3-cephem-4 carboxylic acid or a salt thereof. For example, the electrochemical sensing device could be an electrode, chip, kit, or ironically charged membrane.

The reaction of a lysed sample, with a reagent kit is conducted over a period of time sufficient to observe a color change in the (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 solution when carbapenemase-producing Enterobacteriales, Pseudomonas or Acinetobacter are present in the test sample. Typically, the colour change is observed less than 1 hour and more preferably less than 30 minutes from mixing the sample with the buffer/reagent. Typically, the reaction of the sample with a reagent kit is carried out at a temperature comprised from 5°C to 40°C, preferably from 15°C to 30°C, and more preferably at room temperature.

The reagent kit may be used to provide a very rapid way of identifying an infection with carbapenemase-producing Enterobacteriaceae, Pseudomonas or Acinetobacter bacteria with high reliability and accuracy, according to the method of the present invention.

Figures

Figure 1 shows phenotypic results for Pseudomonas spp. Figure 2 shows phenotypic results for Acinetobacter baumanii.

Figure 3 shows comparative testing of Pseudomonas and Acinetobacter spp. using a method of the present invention and the standard technique.

Figure 4 shows phenotypic results for the detection of carbapenemase producing Enterobacteriales

The invention will further be illustrated in view of the following and examples. Example 1

A test solution comprising an inhibitor mix and ([(7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)- 2(methoxyimino)acetamide]-3-(2,4-dinitrostyryl)-3-cephem-4 carboxylic acid, was produced and used to determine the presence of carbapenemase-producing Pseudomonas or Acinetobacter in a number of tests samples, as set out in Tables 1 and 2.

The test solution was made up as follows:

1) Clavulanic acid and cloxacillin were made up to a final concentration of 40μg/ml and 400μg/ml and mixed together in equal parts to yield the inhibitor mix. 2) (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)aceta mide]-3-(2,4- dinitrostyryl)-3-cephem-4 carboxylic acid or a salt thereof in powder form was dissolved in 1 part DMSO to 7 parts dicarboxylic acid, followed by the addition of zinc sulphate to a final concentration of ImM.

3) A Ιμΐ loopful of either Pseudomonas or Acinetobacter spp was suspended in ΙΟΟμΙ of a commercially available protein extraction buffer/reagent or ΙΟΟμΙ of a extraction buffer comprising of 50mM Tris HC1, lOOmM NaCl, and 1% Triton X100 in a microfuge tube.

4) The suspended Pseudomonas or Acinetobacter spp was mixed by vortexing for a period of 5 seconds prior to heating of the sample in the microfuge to 35°C-37°C and incubating for a period of 10 minutes to produce a lysed sample.

5) 50μ1 of the lysed sample was added to 50μ1 of the (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 solution and ΙΟΟμΙ of the inhibitor mix in a well of a 96-microtitre plate at room temperature.

6) The contents of the well were mixed by pipetting during the addition of the cell lysate.

7) The sample was visually monitored for up to 30 minutes, with the solution colour noted at 5, 10, 20 and 30 minutes from mixing of the sample with the ([(7R)-7-[(z)-2-(2- aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dini trostyryl)-3-cephem-4 carboxylic acid or a salt thereof solution.

Table 1: Panel of Pseudomonas spp and result obtained with candidate chromogenic substrate

10-100μ1 of inhibitors

Result: Red/Orange = +(pos) Yellow =

Carbapenemase -(neg)

Organism ATCC/NCTC

Resistance Time after addition of the substrate,

CX & CLV (minutes)

5 10 20 30

Pseudomonas

No - - - - - aeruginosa Pseudomonas

No - - - - - aeruginosa

Pseudomonas ATCC 27853

No - - - - aeruginosa NCTC 12903

Pseudomonas

No - - - - - aeruginosa

Pseudomonas ATCC 25668

No - - - - aeruginosa NCT 10662

Pseudomonas

No - - - - - aeruginosa

Pseudomonas

No - - - - - spp.

Pseudomonas

No - - - - - spp.

Pseudomonas

No - - - - - spp.

Pseudomonas

No - - - - - spp.

Pseudomonas

NDM-1 - + + + + spp.

Pseudomonas

NDM-1 - + + + + spp.

Pseudomonas

No - - - - - aeruginosa

Pseudomonas

NDM-1 - + + + + aeruginosa

Pseudomonas

No - - - - - aeruginosa

Pseudomonas

No - - - - - aeruginosa

Pseudomonas

IMP-1 - + + + + spp.

Pseudomonas

No - - - - - aeruginosa

Pseudomonas

NDM-1 - + + + + spp.

Pseudomonas

VIM-4 - + + + + aeruginosa

Pseudomonas

VIM-2 - + + + + aeruginosa

Pseudomonas

IMP-1 - + + + + aeruginosa Pseudomonas

No - - - - - aeruginosa

Pseudomonas

No - - - - - aeruginosa

Pseudomonas

VIN-10 NCTC 13437 + + + + aeruginosa

As shown in Table 1, (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 successfully detected all 9 carbapenemase producing Pseudomonas spp. The sensitivity of the method of the present invention was 100 %, as was the specificity.

Corresponding phenotypic results for Pseudomonas spp. are depicted in Figure 1.

These tests show that the method of the present invention is very sensitive for the detection of NDM-1, IMP and VIMs in Pseudomonas spp. Furthermore the yellow colour of the (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)aceta mide]-3-(2,4- dinitrostyryl)-3-cephem-4 carboxylic acid or a salt thereof solution is shown to undergo a significant change to a red colour within 5 minutes after the addition of lysed bacterial samples at room temperature. While a stronger red may develop over longer times, the colour change after 5 minutes is enough to show a positive reaction.

Table 2: Panel of Acinetobacter spp and result obtained with candidate chromogenic substrate

10-100μ1 of inhibitors

Result: Red/Orange = +(pos) Yellow =

Carbapenemase -(neg)

Organism ATCC/NCTC

producer Time after addition of the substrate,

CX & CLV (minutes)

5 10 20 30

Acinetobacter

No - - - - - spp.

Acinetobacter ACTC 15309

No - - - - spp. NCTC 5866

Acinetobacter

No - - - - - spp.

Acinetobacter

No - - - - - baumanii Acinetobacter

No - - - - - spp.

Acinetobacter

No - - - - - spp.

Acinetobacter

No - - - - - spp.

Acinetobacter

No - - - - - spp.

Acinetobacter

No - - - - - haemolyicus

Acinetobacter

No - - - - - Iwoffii

Acinetobacter

No - - - - - baumanii

Acinetobacter

No - - - - - baumanii

Acinetobacter

MBL - + + + + baumanii

Acinetobacter

No - - - - - spp.

Acinetobacter

No - - - - - spp.

Acinetobacter

No - - - - - spp.

Acinetobacter

No - - - - - spp.

Acinetobacter

OXA-23 NCTC 13301 + + + + baumanii

Acinetobacter

OXA-25 NCTC 13302 + + + + baumanii

Acinetobacter

OXA-26 NCTC 13303 + + + + baumanii

Acinetobacter

OXA-27 NCTC 13304 + + + + baumanii

Acinetobacter

OXA-58 NCTC 13305 + + + + baumanii

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 a salt thereof successfully detected all 5 carbapenemase producing Acinetobacter baumanii. The sensitivity of the method of the present invention was 100 %, as was the specificity.

Corresponding phenotypic results for Acinetobacter baumanii are depicted in Figure 2.

These tests show that the method of the present invention is very sensitive for the detection of OXAs in Acinetobacter spp. Furthermore the yellow colour of the (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 solution is shown to undergo a significant change to a red colour within 5 minutes after the addition of lysed bacterial samples at room temperature. While a stronger red may develop over longer times, the colour change after 5 minutes is enough to show a positive reaction.

Example 2

A test solution was made up according to the method of Example 1 and was used to test five Pseudomonas isolates; of which 4 isolates were resistant to the carbapenems and 1 was a carbapenemase non-producer. Of the 4 carbapenemase producing Pseudomonas isolates, 2 were DM-ls, whilst the remaining 2 were VIMs. Six Acinetobacter isolates were also tested; of which 5 isolates were resistant to the carbapenems and 1 was a carbapenemase non-producer. All the 5 carbapenemase producing Acinetobacter isolates were OXAs.

Corresponding tests for each Pseudomonas and Acinetobacter isolate were also tested using the commercially available colorimetric test for detecting strains with reduced susceptibility to the carbapenems, known to the skilled person in the field. Following standard instruction, the comparative tests colorimetric based testing technique were performed at 35°C - 37°C, while the tests carried out according to a method of the present invention were performed at room temperature.

The results of these test are depicted in Table 3 with the observed colour changes shown in Figure 3.

The test included a negative control Pseudomonas and Acinetobacter isolate consisting of a carbapenemase negative Pseudomonas aeruginosa and an Acinetobacter iwoffi isolate. All of the remaining isolates were carbapenemase producers. Table 3: Comparison of test solution mix comprising of (7R)-7-[(z)-2-(2- aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dini trostyryl)-3-cephem-4 carboxylic acid or a salt thereof, versus a commercially available colori eiric jest for detecting strains with reduced susceptibility to the carbapenems

As shown in Table 3, of the 11 organisms tested during the comparison of (7R)-7-[(z)-2- (2-aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-d initrostyryl)-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)aceta mide]-3-(2,4- dinitrostyryl)-3-cephem-4 carboxylic acid or a salt thereof, successfully detected all nine of the carbapenemase producing Pseudomonas and Acinetobacter isolates, while tests using the standard technique did not detect carbapenemase production in one Acinetobacter isolate (highlighted in table). The false negative that was misidentified using the standard test, was an OXA-27.

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

Example 3.

Two test solutions were made up according to the method of Example 1. One of the solutions was exactly as described in Example 1 and the other solution utilized the chromogenic cephalosporin HMRZ-86 in place 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 the carbapenems and 60 were carbapenemase non-producers. Of the 8 carbapenemase producing Pseudomonas isolates, 4 were DM-ls, 2 were VIMs, while the remaining 2 were EVIPs.

Both test solutions either using HMRZ-86 or (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)- 2(methoxyimino)acetamide]-3-(2,4-dinitrostyryl)-3-cephem-4 carboxylic acid, were carried out according to a method of the present invention (performed at room temperature). The results of the tests are depicted in Table 4 Table 4: Comparison of the sensitivity of (7R)-7-[(z)-2-(2-aminoth iazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dinitrostyr yl)-3- cephem-4 carboxylic acid or a salt thereof versus HMRZ-86, for the detection of carbapenemase producing Pseudomonas using the present invention setup

ΙΟΟμΙ of inhibitors (HM RZ-98) ΙΟΟμΙ of inhibitors (HM RZ-86)

Result: Red/Orange = +(pos) Yellow = -(neg) Result: Red/Orange = +(pos) Yellow = -(neg)

Time after addition of HMRZ98, CX400, CLV40 Time after addition of HMRZ86, CX400, CLV40

Carbapenemas ATCC/NCT 5 10 20 30 5 10 20 30

Organism e Resistance C Minutes Minutes Minutes Minutes Minutes Minutes Minutes Minutes

Pseudomonas spp. No - - - - - - - - -

Pseudomonas

aeruginosa No

Pseudomonas

aeruginosa No

Pseudomonas

fluorescens No

Pseudomonas

aeruginosa No

Pseudomonas

aeruginosa No

ATCC

25668

Pseudomonas NCTC

aeruginosa No 10662

Pseudomonas

aeruginosa No

Pseudomonas spp. No - - - - - - - - -

Pseudomonas

aeruginosa No

Pseudomonas spp. No - - - - - - - - -

Pseudomonas No - - - - - - - - -

aeruginosa

Pseudomonas spp. No - - - - -

Pseudomonas spp. No - - - - -

Pseudomonas spp. No - - - - -

Pseudomonas spp. No - - - - -

Pseudomonas spp. No - - - - -

Pseudomonas spp. No - - - - -

Pseudomonas spp. No - - - - -

Pseudomonas spp. No - - - - -

Pseudomonas

aeruginosa No

Pseudomonas spp. No - - - - -

Pseudomonas

aeruginosa No

Pseudomonas

aeruginosa No

Pseudomonas spp. No - - - - -

Pseudomonas

aeruginosa No

Pseudomonas spp. No - - - - -

Pseudomonas spp. No - - - - -

Pseudomonas spp. No - - - - -

Pseudomonas spp. No - - - - -

Pseudomonas spp. No - - - - -

Pseudomonas spp. No - - - - -

Pseudomonas spp. No - - - - -

Pseudomonas spp. No - - - - -

Pseudomonas spp. No - - - - -

Pseudomonas spp. No - - - - -

Pseudomonas spp. No

Pseudomonas spp. NDM-1

Pseudomonas spp. NDM-1

Pseudomonas

aeruginosa No

Pseudomonas spp. No

Pseudomonas

aeruginosa No

Pseudomonas

aeruginosa NDM-1

Pseudomonas

aeruginosa No

Pseudomonas

aeruginosa No

Pseudomonas

aeruginosa No

Pseudomonas

aeruginosa No

Pseudomonas

aeruginosa No

Pseudomonas

aeruginosa No

Pseudomonas

aeruginosa No

Pseudomonas

aeruginosa IMP-1 Contaminated Pl.ite

Pseudomonas No

aeruginosa

Pseudomonas spp. NDM-1 + + + +

Pseudomonas

aeruginosa No

Pseudomonas

aeruginosa VI M-4 + + + +

Pseudomonas

aeruginosa VI M-2 + + + +

Pseudomonas

aeruginosa No

Pseudomonas

aeruginosa I M P-1 + + + +

Pseudomonas

aeruginosa No

Pseudomonas

aeruginosa No

Pseudomonas

aeruginosa No

Pseudomonas aureus No - - - - -

Pseudomonas

aeruginosa No

Pseudomonas

aeruginosa No

Pseudomonas

aeruginosa No

Pseudomonas

aeruginosa No

Pseudomonas spp. No - - - - -

Pseudomonas spp. No - - - - -

As shown in Table 4, of the 8 carbapenemase producing Pseudomonas organisms tested during the comparison of (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)- 2(methoxyimino)acetamide]-3-(2,4-dinitrostyryl)-3-cephem-4 carboxylic acid, versus HMRZ, (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)aceta mide]-3-(2,4- dinitrostyryl)-3-cephem-4 carboxylic acid, successfully detected all eight of the carbapenemase producing Pseudomonas isolates, while tests using the HMRZ-86 in place of (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)aceta mide]-3-(2,4- dinitrostyryl)-3-cephem-4 carboxylic acid did not detect two DM-1 producing isolates (highlighted in table 4).

Accordingly, it is shown that the present invention technique has a sensitivity and specificity of 100%, whilst the candidate chromogenic substrate with HMRZ-86 results in a 75%) sensitivity. The method of the present invention with (7R)-7-[(z)-2-(2- aminothiazol-4-yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dini trostyryl)-3-cephem-4 carboxylic acid therefore boasts higher sensitivity for the rapid detection of carbapenemase-producing Pseudomonas or Acinetobacter in test samples,

Example 4

A dicarboxylic acid buffered test solution comprising ([(7R)-7-[(z)-2-(2-aminothiazol-4- yl)-(Z)-2(methoxyimino)acetamide]-3-(2,4-dinitrostyryl)-3-ce phem-4 carboxylic acid, an inhibitor mix, zinc sulphate and a bulking agent, was produced and used to determine the presence of carbapenemase-producing Enterobacteriales, Pseudomonas or Acinetobacter in 147 test isolates, as set out in Table 5.

The test solution was made up as follows:

1) (7R)-7-[(z)-2-(2-aminothiazol-4-yl)-(Z)-2(methoxyimino)aceta mide]-3-(2,4- dinitrostyryl)-3-cephem-4 carboxylic acid, in powder form was dissolved in 1 part DMSO to 7 parts dicarboxylic acid,

2) The solution from step 1 above was then buffered via the additional supplementation with a dicarboxylic acid in the ratio of 1 part test solution to 4 parts dicarboxylic acid. Clavulanic acid and cloxacillin were made up to a final concentration of 40μ§/ιη1 and 400μ§/ιη1 and mixed together in equal parts to yield the inhibitor mix.

Zinc sulphate and PEG 8000 were added to the inhibitor mix, to final concentrations of ImM and 0.5% respectively.

A mixture containing 2mM polymixin, 4mM of a glycopeptide and 1.5mM of a polypeptide antibiotic were also dissolved in the solution containing the inhibitor mix

2.8ml volumes solution containing the inhibitor mix, zinc sulphate, PEG 8000 and antibiotic mixture described in step 5 above were then freeze dried into pellets for 72hr

Subsequently, determining the presence of a carbapenemase was then carried out as described below:

(a) Reconstitute the pellet by adding 3.5ml of test solution from step 2 above.

(b) Allow the pellet to fully dissolve at room temperature for 1 minute and mix contents by gently vortexing for 10 seconds.

(c) Reconstituted solution should be yellow, if the solution is any other colour do not use.

(d) Dispense 500μ1 of reconstituted solution into microfuge tubes. One tube per test.

(e) Using a pure, fresh culture of the test organism, add a Ι μΐ loopful of organism to the microfuge tube and mix well by vortexing for 10 seconds.

(f) Incubate at 35-37°C for 10 minutes and record the colour of the test solution Table 5: External and internal evaluation data showing high senstivity and specificity for the detection of carbapenemase producing Enterobacteriales, Pseudomonas and Acinetobacter spp isolates, with the present invention.

Correctly Identified Incorrectly Identified

Resistance Mechanisms

(Carbapenemase (Carbapenemase Negative) Positive)

ESpL 26 0

AmpC 17 0

Non-Carbapenemase

AmpC/PL 6 0

Producing Organisms

Negative 15 0

Total 64 0

Correctly Identified Incorrectly Identified

Resistance Mechanisms

(Carbapenemase (Carbapenemase Positive) Negative)

KPC 14 1

MpL 39 0

OXA-48 16 0

OXA-48 & ESpL 1 0

OXA-95 & AmpC 0 1

OXA-23 4 0

Carbapenemase Producing OXA-25 1 0

Organisms

OXA-26 1 0

OXA-27 1 0

OXA-23 & OXA-51 2 0

OXA-58 1 0

OXA-23 & OXA-27

1 0

& OXA-51

Total 81 2 Sensitivity 97.59%

Specificity 100.00%

1 10 Enterobacteriales were tested; of which 72 isolates were carbapenemase producers and 38 were non-carbapenemase producers. 57 of the tested isolates were Klebsiella spp [12 ESBLs, 5 AmpCs, 10 OXAs, 13 KPCs and 17 Μβίε (6 NDMs, 6 IMPs and 5 VIMs)], 24 were Escherichia coli [8 ESBLs, 5 AmpCs, 5 OXAs, 1 KPC and 5 Μβίε (2VIMs, 2NDMs and 1 IMP)], 23 were Enterobacter spp [6 ESBLs, 10 AmpCs, 2 OXAs, 4 Μβίε (2 VIMs, 2 NDMs and 1 KPC)], 3 Citrobacter spp [all NDMs], 1 Kluvyera spp [an NDM producer] and 2 Salmonella spp isolates were tested; of which both were AmpC producers [non- carbapenemases]

23 Pseudomonas spp isolates were tested; of which 8 isolates were MpL carbapenemase producers comprising of 2 NDMs, 2 IMPs and 4 VIMs. 15 Pseudomonas isolates were carbapenemase non-producers [14 of these did not have any enzyme mediated mechanism, while one isolate was an AmpC producer].

14 Acinetobacter spp isolates were tested; of which 13 isolates were carbapenemase producers and one was a carbapenemase non-producer. Of the carbapenemase producers, there were 12 oxacillinase producing isolates [4 OXA-23s, 1 OXA-25, 1 OXA- 26, 1 OXA-27, 1 OXA-58 and co-producers such as 2 OXA23 + OXA-51 s, 1 OXA-23 + OXA-27 + OXA-51 and 1 OXA-95+AmpC]. One of the carbapenemase producing Acinetobacter isolates was an MpL producer.

As shown in Table 5, of the total 147 isolates tested, the present invention detected 81 of the 83 carbapenemase producing Enterobacteriales, Pseudomonas and Acinetobacter isolates. This correlates to a total sensitivity of 97.59% and 100% specificity for a wide range of Gram negative bacteria. Meanwhile a commercially available kit has been judged to be not suitable for use with Pseudomonas aeruginosa (Henrichs et a/., 2015), whilst another major competitor kit which is believed to employ the use of the chromogenic HMRZ-86 only showed a 64.9% sensitivity and 90% specificity for the detection of carbapenemase producing Enterobacteriaceae (Mancini et a/., 2017). Consequently, at this moment, the present invention is the sole rapid colorimetric carbapenemase detection method that is not only suitable for use with Pseudomonas and Acinetobacter spp isolates, but also extends to the Enterobacteriales; with very high sensitivity and specificity.