Method and device for the detection of carbapenemases and carbapenemase producers

Abstract

The invention refers to a method and a device for the phenotypic detection of carbapenemases and carbapenemase producers by adding a substrate of general formula A-(L)-M.sub.1-(X)—Z, where M.sub.1 is a carbapenem backbone, A or Z is a quencher, the other one of the two, Z or A, is a fluorophore, L is an optional linker, X is an optional leaving group for linking Z to the carbapenem backbone, and Z is an optional leaving group, to a sample suspected of containing such carbapenemase producers and/or carbapenenmases. The invention further refers to a method for the phenotypic detection of resistant bacteria, in particular 3MRGN or 4MRGN, by releasing the enzymes of a bacterial culture into a lysate during lysis and then subjecting the lysate, as the sample to be analyzed, to an aforementioned method in order to phenotypically detect the presence of resistance-conferring carbapenemases.

Claims

1. A method for the phenotypic detection of carbapenemase and carbapenemase producers, the method comprising: in a medium contacting a substrate of structural formula (I) with a sample suspected of containing the carbapenemases and/or carbapenemase producers: ##STR00014## wherein R1 is absent or selected from the group consisting of —CH.sub.2—, —O—, —S—, —CO—, —CHOH—, and (—CH═CH).sub.n—CH.sub.2—; R2 is selected from the group consisting of —H, —OH, —SH, halogen, a C1-C6 alkyl optionally substituted with —OH, ═O, —NH2, ═S, or —SH, an optionally substituted oxyalkyl, an optionally substituted alkenyl, an optionally substituted aromatic compound, an optionally substituted heterocyclic compound, a primary amine, a secondary amine, an ester, an ether, thiol, and a thioether; R is selected from the group consisting of a C1-C6 alkyl optionally substituted with —OH, ═O, —NH2, ═S, or —SH, an optionally substituted oxyalkyl, an optionally substituted alkenyl, an optionally substituted aromatic compound, an optionally substituted heterocyclic compound, a secondary amine, an ester, an ether, and a thioether; n is an integer from 1 to 4; (L) is an optional linker wherein i) (L) is selected from the group consisting of a C.sub.1-C.sub.12 alkyl, C.sub.1-C.sub.12 oxyalkyl, C.sub.1-C.sub.12 alkene, and C.sub.1-C.sub.12 alkyne, each optionally substituted by —OH, —SH, ═O, —NH.sub.2, ═S, halogen, a primary amine, a second amine, an ester, an ether and a thiol; or ii) (L) is selected from the group consisting of a thioether, an aromatic compound, a cyclic compound, an ester, an ether, a carbamate, and an amide; (X) is an optional group, wherein i) (X) is selected from the group consisting of a —S—R—, a sulphonate, a substituted tosylate, a mesylate, a thiophenolate, a substituted thiophenolate, —S-aryl-, a phenolate, a substituted phenolate, and —O-aryl-; or ii) (X) comprises a carbamate, a carbonate, a thiocarbamate, a thiocarbonate, an inorganic ester, an organic ester, an anhydride, an alcohol, a carboxylic acid, a halide, and diazo; or iii) (X) is —S—, —O—SO.sub.2), or —O—SO.sub.2—R—; A and Z form a fluorophore/quencher pair, or A represents a reporter group and Z represents a solid phase, or A represents a solid phase and Z represents a reporter group, wherein when A and Z form a fluorophore/quencher pair, then A is a quencher and Z is a fluorophore, or A is a fluorophore and Z is a quencher, and wherein if (X) is present, then the group X—Z is released after enzymatic cleavage of the substrate by carbapenemase, or if (X) is absent, then the group Z is released after enzymatic cleavage of the substrate by carbapenemase; and detecting the change in fluorescence intensity observed after the separation of the fluorophore from the quencher following cleavage of the substrate by the carbapenemase; or detecting the reporter group cleaved from or with the substrate following cleavage of the substrate by the carbapenemase.

2. The method according to claim 1, wherein R2 is H or methyl.

3. The method according to claim 1, wherein R1 is selected from the group consisting of —CH.sub.2—, —O—, —S—, (—CH═CH).sub.n—CH.sub.2—, and —CO—.

4. The method of claim 3, wherein R1 is —CH.sub.2—.

5. The method according to claim 1, wherein R2 is —H or methyl.

6. The method according to claim 1, wherein prior to the release of the fluorophore or quencher, the fluorescence of the fluorophore is reduced or quenched by Forster resonance energy transfer to the quencher and after the release of the fluorophore or the quencher, the quencher and the fluorophore are spatially separated from each other so that no more Forster resonance energy transfer takes place and a change in the fluorescence is measured.

7. The method according to claim 1, wherein the fluorophore is used as quencher, which acts as fluorescence acceptor in the Förster resonance energy transfer, or wherein a non-fluorescent quencher, which is energetically adapted to the fluorophore and extinguishes the fluorescence of the fluorophore in the bound state, is used as quencher.

8. The method according to claim 1, wherein the fluorescence intensity of the fluorophore reacts insensitively to pH changes of the medium.

9. The method according to claim 1, wherein the fluorophore is a self-quenching fluorophore.

10. The method according to claim 1, wherein the reporter group is selected from the group consisting of a quencher, a fluorophore, a chromogenic group, an absorbent dye, an azo dye, a metal complex dye, a dioxazine dye, an indigo dye, a nitro and/or sulfur dye, a triphenylmethane dye, a phthalocyanine dye, a nitroso dye and a radioactively labeled group.

11. The method according to claim 1, wherein the linker (L) used is a linker which is or contains an ether, an ester, a carbamate, or an amide.

12. The method according to claim 1, wherein group (X) is —S—, —O—SO2—, or —O—SO2—R—.

13. The method according to claim 1, wherein the sample additionally contains a substrate of structural formula (IV) ##STR00015## wherein after enzymatic cleavage of the substrate by beta-lactamase the group Z is released, wherein A, (L), R1, (X) and Z have the same meanings as in claim 1.

14. The method according to claim 13, wherein the substrates (I) and (IV) have distinct reporter groups to phenotypically detect carbapenemase and beta-lactamase producers or the enzymes carbapenemase and extended spectrum beta-lactamase (ESBL) in the sample in parallel and simultaneously.

15. The method according to claim 13, wherein the substrates (I) and (IV) have fluorophores with different emission maxima.

16. A method for phenotypically detecting resistant bacteria, the method comprising: releasing the enzymes of a bacterial culture into a lysate; and subsequently subjecting the lysate as a sample to be investigated to the method according to claim 1 to phenotypically detect the presence of resistance-conferring carbapenemases or extended spectrum beta-lactamases, and thereby detect the presence of resistant bacteria.

17. The method of claim 16, wherein at least one of the following is true: a) the bacteria are multi-resistant Gram-positive or Gram-negative rod bacteria; b) the bacteria are multi-resistant Gram-negative rod bacteria resistant to three or four groups of antibiotics selected from the group consisting of acylaminopenicillins, 3rd and 4th generation cephalosporins, carbapenems, and quinolones; c) the bacterial culture comprises a lysis buffer; or d) lysis of the bacterial culture comprises subjecting the bacterial culture to a mechanical digestion method.

18. The method of claim 17, wherein the mechanical digestion method is ultrasound.

19. The method-according to claim 16, wherein the bacterial culture is first placed in a selective detection medium in which all non-resistant bacteria are killed.

20. The method according to claim 16, wherein the bacteria are lysed after reaching a sufficient cell number, which is determined by measuring the optical density or by a dynamic measurement of the scattered light.

21. A substrate for phenotypic detection of carbapenemases and carbapenemase producers of the structure of formula (I): ##STR00016## wherein R1 is absent or selected from the group consisting of —CH.sub.2—, —O—, —S—, —CO—, —CHOH—, and (—CH═CH).sub.n—CH.sub.2—; R2 is selected from the group consisting of —H, —OH, —SH, halogen, a C1-C6 alkyl optionally substituted with —OH, ═O, —NH.sub.2, ═S, or —SH, an optionally substituted oxyalkyl, an optionally substituted alkenyl, an optionally substituted aromatic compound, an optionally substituted heterocyclic compound, a primary amine, a secondary amine, an ester, an ether, thiol, and a thioether; n is an integer from 1 to 4; (L) is an optional linker wherein R is selected from the group consisting of a C1-C6 alkyl optionally substituted with —OH, ═O, —NH2, ═S, or —SH, an optionally substituted oxyalkyl, an optionally substituted alkenyl, an optionally substituted aromatic compound, an optionally substituted heterocyclic compound, a secondary amine, an ester, an ether, and a thioether; i) (L) is selected from the group consisting of a C.sub.1-C.sub.12 alkyl, C.sub.1-C.sub.12 oxyalkyl, C.sub.1-C.sub.12 alkene, and C.sub.1-C.sub.12 alkyne, each optionally substituted by —OH, —SH, ═O, —NH.sub.2, ═S, halogen, a primary amine, a secondary amine, an ester, an ether, or a thiol; or ii) (L) is selected from the group consisting of a thioether, an aromatic compound, a cyclic compound, an ester, an ether, a carbamate, and an amide; (X) is an optional group, wherein i) (X) is selected from the group consisting of a —S—R—, a sulphonate, a substituted tosylate, a mesylate, a thiophenolate, a substituted thiophenolate, —S-aryl-, a phenolate, a substituted phenolate, and —O-aryl-; or ii) (X) comprises a carbamate, a carbonate, a thiocarbamate, a thiocarbonate, an inorganic ester, an organic ester, an anhydride, an alcohol, a carboxylic acid, a halide, and diazo; or iii) (X) is —S—, —O—SO.sub.2), or —O—SO.sub.2—R—; A and Z form a fluorophore/quencher pair, or A represents a reporter group and Z represents a solid phase, or A represents a solid phase and Z represents a reporter group, wherein when A and Z form a fluorophore/quencher pair, then A is a quencher and Z is a fluorophore, or A is a fluorophore and Z is a quencher.

22. The substrate of claim 21, wherein group (X) is —S—, —O—SO.sub.2—, or —O—SO.sub.2—R—.

23. A solid phase comprising the substrate of claim 22 bonded to its surface.

Description

SHORT DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the reaction step of a fluorescence-based method for the detection of extended spectrum β-lactamase (ESBL) in a sample.

(2) FIG. 2 shows the reaction step of a fluorescence-based method for the detection of carbapenemase in a sample according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

(3) The phenotypic detection of a (β-lactam ring cleaving enzyme such as a carbapenemase or an ESBL requires the synthesis of a specific labelled substrate that can be cleaved by such an enzyme and, after cleavage, triggers a measurable signal. This signal can either be a released reporter group, or it can be an optical signal. In order to trigger a measurable optical signal, a substrate labelled with a fluorophore and an energetically suitable quencher (in particular a Black Hole Quencher, BHQ) is used, which can be cleaved by such an enzyme. The selection of suitable fluorophore/quencher pairs is known to the person skilled in the art (Fluorescent Energy Transfer Nucleic Acid Probes, Designs and Protocols; Editors: Vladimir V. Didenko M D, PhD, (2006), Humana Press). The detection reaction obeys the following general relationship

(4) ##STR00005##
wherein Q denotes a quencher group, Fl denotes a fluorophore group, M.sub.1 denotes a compound containing an β-lactam ring, and M.sub.2 denotes a degradation compound after enzymatic cleavage of the compound M.sub.1 by an enzyme E cleaving the β-lactam ring. The connections between Q and M.sub.1 and between Fl and M.sub.1, executed as dashes, can represent or contain linkers and/or leaving groups.

(5) In the method according to the invention for the phenotypic detection of carbapenemases and carbapenemase producers, a sample suspected of containing such carbapenemase producers and/or carbapenemases is provided with at least one substrate of the general structural formula (I)
A-(L)-M.sub.1-(X)—Z  (I)
wherein M.sub.1 is a carbapenem backbone and wherein either A and Z form a fluorophore/quencher pair, or A represents a reporter group and Z represents a solid phase, or A represents a solid phase and Z represents a reporter group, L is an optional linker for coupling A to the carbapenem backbone, and X is an optional leaving group for linking Z to the carbapenem backbone. After enzymatic cleavage of the substrate by carbapenemase the group Z is released (depending on the reaction together with, or separated from, the leaving group X).

(6) The carbapenem backbone M.sub.1 is preferably a compound of formula (M.sub.1)

(7) ##STR00006##

(8) wherein R, R1 and R2 are each independently H, a C.sub.1-C.sub.6 alkyl, (methyl, ethyl, propyl, butyl, t-butyl, isopropyl, optionally substituted with e.g. —OH, —SH═O, ═S, —NH.sub.2), optionally substituted oxyalkyl, a halogen, —OH, —SH, a primary or secondary amine, an ether, an ester, an optionally substituted alkenyl, an optionally substituted aromatic compound, an optionally substituted heterocyclic compound, a thiol or a thioether. In a particularly preferred embodiment, R2 is either H or methyl. Preferably R1 is —CH.sub.2—, —O or —CO—, especially preferably R1 is CH.sub.2.

(9) In an embodiment, the group A-(L)- is linked to the beta-lactam ring of the carbapenem backbone and the group —(X)—Z is linked to the five-membered ring of the carbapenem backbone. Preferably the group A-(L)- at the position of substituent R is linked to the carbapenem backbone (R=A-(L)-) or is attached to the substituent R and the group —(X)—Z is preferably linked to the carbapenem backbone at the position of substituent R1 (R1=—(X)—Z) or is attached to the substituent R1.

(10) If no fluorophhore/quencher pair is used, the reporter group (A or X) is selected from the group consisting of a quencher, a fluorophore, a chromogenic group, an absorbent dye such as an azo dye, a metal complex dye, a dioxazine dye, an indigo dye, a nitro and/or sulfur dye, a triphenylmethane dye, a phthalocyanine dye, a nitroso dye (having a chromophoric group such as R—C═O—R, R—N═N—R, R—NO.sub.2, R—O═O, R—C═NH, R—N═O); a radioactively labelled group (e.g, by using isotopes: e.g. 3H, 11C, 13C, 14C, 13N, 15O, 18F, 26Al, 32P, 33P, 35S, 36Cl, 41Ca, 125I, 131I).

(11) If a solid phase is used, i.e. the substrate is immobilized via a linker to a surface, the surface can be a glass substrate, a film substrate (substrates can be e.g. PEN, PET), PDMA, or particulate structures (nanoparticles, microparticles e.g. silica particles, beads e.g. magnetic beads) or a polymer (e.g. polystyrene). Immobilization via the linker (L) can be achieved by chemisorption (Au—HS—R), by covalent bonding (e.g. via aldehyde, epoxy, isothiocyanate functionalization of the surface, these react with H2N—R; via maleimide, mercaptosilane functionalization of the surface, this binds thiols), or by non-covalent bonding (e.g. via biotin-R to streptavidin-functionalized surface (and vice versa)).

(12) Suitable linkers (L) are e.g. C.sub.1-C.sub.2 alkyl and oxyalkyl (methyl, ethyl, propyl, butyl, t-butyl, iso-propyl, pentyl, hexyl, heptyl octyl, nonyl, decyl, undecyl, dodecy, in addition to alkanes also alkenes and alkynes, optionally substituted with e.g. —OH, —SH, ═O, NH.sub.2, ═S, halogen, primary or secondary amines, esters, ethers, thiols, further they may consist of/include: thioethers, aromatic compounds, cyclic compounds and heterocycles. Linkers can be equipped with functional groups for connection to a fixed phase, e.g. carboxylic acids, thiocarboxylic acids, peroxycarboxylic acids, thiols, sulphonic acids, sulphinic acids, sulphoxides, carboxylic acid anhydrides, carboxylic acid esters, sulphonic acid esters, nitric acid esters, carboxylic acid halides, sulphonic acid halides, carboxylic acid amides, sulphonic acid amides, carboxylic acid hydrazides, nitriles, aldehydes, thioaldehydes, ketones, thioketones, oximes, hydrazones, alcohols, phenols, amines, imines, hydrazines, thioethers, thiol esters and ethers, as well as their activating forms (e.g. carboxylic acid chlorides, NHS esters, . . . ), the linker can also be biotinylated for binding to streptavidin (and vice versa).

(13) Suitable chemical leaving groups (X) are e.g. thiols —S—R, sulphonates, substituted tosylates, and mesylates, thiophenolates and substituted thiophenolates, —S—Ar, phenolates or substituted phenolates, phenoxides —O—Ar (where Ar=aryl). In addition, the leaving groups may be derivatives of carbamates, carbonates, thiocarbamates or thiocarbonates, inorganic esters (e.g. of phosphoric acid/phosphates or sulphuric acid/sulphonates), organic esters, anhydrides, alcohols, carboxylic acids and halides (e.g. I— for radioactive labels) and diazo compounds. A preferred leaving group (X) is a leaving group which is or contains a thioether —S— or a thioether compound —S—R— or a sulphonic acid ester (—O—SO.sub.2—) or a sulphonic acid ester compound (—O—SO.sub.2—R).

(14) In a preferred embodiment of the method according to the invention the phenotypic detection of carbapenemase or carbapenemase producers, a sample suspected of containing carbapenemase or carbapenemase producers is provided with a substrate of the general structural formula (III)

(15) ##STR00007##

(16) wherein R1 is absent or selected from the group consisting of —CH.sub.2—, —S—, —CO—, CHOH, (—CH═CH).sub.n CH.sub.2− (where n=1-4), and R2, A, L, X and Z are as defined above. In particularly preferred embodiments, R2 is methyl and R1 is a CH.sub.2 group, R2 is methyl and R1 is absent, R2 is H and R1 is a CH.sub.2 group, or R2 is H and R1 is absent. Especially preferred substrates are:

(17) ##STR00008##

(18) After enzymatic cleavage of the above substrate by carbapenemase, the above substrate (III) decomposes into the reaction products

(19) ##STR00009##

(20) Depending on the embodiment for X or Z, X can also be released in addition to Z, or the group —(X)—Z can be released.

(21) The detection method is based on a change in fluorescence observed after the separation of the fluorescent group Z or A from the corresponding quenching group A or Z, or on the detection of the reporter group cleaved from or with the substrate. The process is per se specific for carbapenemases which accept the respective substrate and hydrolytically open the β-lactam ring to form a carboxylic acid and simultaneously release the fluorophore or quencher (Z) or reporter group. If a fluorophore/quencher pair is used, the release of the fluorophore or quencher and the associated increase in fluorescence, for example, can be attributed to the presence of a carbapenemase cleaving the β-lactam ring of the carbapenem in the respective sample. If the fluorophore/quencher pair is a FRET pair, a fluorescence decrease and/or a shift of the fluorescence wavelength can be measured if one of the two partners is split off. The only factor for the release of the fluorophore, quencher or reporter group is the enzyme activity. Thus, a measured change in fluorescence or the detection of the released reporter group is a direct and reliable measure of the presence of carabapenemase in the sample. If a substrate with one of the other reporter groups described above (e.g. dye, radioactive group) is used, the detection can be performed in a manner suitable for the respective reporter group (detection of radioactivity released by the solid phase, UV Vis spectroscopic measurement, etc.). As a non-limiting example, the substrate can be coupled to a solid phase (A), where A is a bead or a glass surface. Under the influence of a sample containing a carbapenemase, the reporter group (Z) is separated from the solid phase and can be detected in the supernatant, or a de-coloration of the beads can be observed. Another example is a test strip on which the substrate(s) according to the invention has (have) been immobilized, where de-coloration of a band with immobilized substrate and/or release of the reporter group is detected (e.g. by paper chromatographic detection of the released (chrornophoric) reporter group). These embodiments make it possible to provide proof by simple means, so that it can be reliably carried out even under difficult conditions, e.g. in areas with a lower development standard.

(22) By using a fluorescence measurement system in combination with the fluorescence-based enzymatic detection reaction described above, the time from sampling to obtaining a result on the presence of carbapenemase in the sample is enormously reduced. Thus, the method according to the invention offers the possibility of rapid on-site detection of carbapenemase in the sample.

(23) In a particularly preferred version of the invention, the sample can be provided with both the aforementioned substrate for fluorescence-based detection of carbapenemase and the aforementioned substrate for fluorescence-based detection of β-lactamase such as ESBL of the general structural formula (IV)

(24) ##STR00010##
wherein R1, A, (L), (X) and Z are as defined above. After enzymatic cleavage by β-lactamase, the above substrate is metabolized into

(25) ##STR00011##

(26) Depending on the embodiment for X or Z, X can also be released in addition to Z, or the group —(X)—Z can be released.

(27) The substrate used to detect the enzyme β-lactamase is based on a cephalosporin backbone which has been chemically modified by covalent binding of a quencher and a fluorophore group or another reporter group. By integrating the fluorescent or quenching group or the reporter group —(X)—Z, the fluorophore or the quencher or the reporter group (Z) is released after enzymatic cleavage of the β-lactam ring and a change in the fluorescence intensity can be detected ora detection of the free reporter group is possible.

(28) In such an embodiment, in which the aforementioned substrate for fluorescence-based detection of β-lactamase and the aforementioned substrate for the fluorescence-based detection of carbapenemase are added to the sample, fluorophores with different emission maxima are bound to the two substrates (i.e. on the one hand to the substrate for the detection of β-lactamase and on the other hand to the substrate for the detection of carbapenemase) in order to be able to carry out the phenotypic detection of β-lactamase and carbapenemase in the sample simultaneously and in parallel.

(29) Due to the different fluorescence emissions of the two fluorophores, the proportion of fluorophore released from the first substrate cleaved by carbapenemase and the proportion of fluorophore released from the second substrate cleaved by β-lactamase can be clearly determined and thus assigned to the respective enzymes.

(30) Alternatively, the reporter groups and/or the fluorophore/quencher pairs can be selected to allow differential detection of the reporter groups and/or fluorophore/quencher pairs of the different substrates (e.g.: combination of a fluorophore/quencher pair for carbapenemase substrate (III) and a chromogenic reporter group/solid phase for substrate (IV), or use of two different dyes e.g. to allow the use of both substrates on one test strip).

(31) Preferably, prior to the release of the fluorophore or the quencher (Z), the fluorescence of the fluorophore is reduced or extinguished by Förster resonance energy transfer to the quencher and after the release of the fluorophore or the quencher (Z) the quencher and the fluorophore are spatially separated from each other so that no more Förster resonance energy transfer takes place and a change in fluorescence is measured.

(32) Through the Förster resonance energy transfer, the energy in the substrate is transferred from the excited fluorophore via dipole-dipole interactions to the quencher. The transferred energy is therefore no longer available to the fluorophore for direct radiation emission. In the presence of the β-lactam ring cleaving enzyme, however, the fluorophore is separated from the quencher with a simultaneous increase in fluorescence.

(33) Another preferred embodiment provides that the quencher itself is a fluorophore. Thus two fluorophores (a reporter fluorophore and a quencher fluorophore) bind to the substrate. In this bound state the emission of the reporter is quenched. However, the enzymatic cleavage of the substrate leads to a spatial separation of reporter fluorophore and quencher fluorophore and thus to a clear signal increase in the receiver unit for the absorption of luminescent radiation. By measuring the signal increase, the respective enzyme (carbapenemase and possibly extended spectrum β-lactamase) can be detected very sensitively. Examples of fluorophore-fluorophore-pairs suitable for enzyme detection are: Fluorophore 1: Cy2-Fluorophore 2: Cy3, Fluorophore 1: Carboxyfluorescein-Fluorophore 2: Texas Red, Fluorophore 1: Fluorescein-Fluorophore 2: Rhodamine.

(34) Another preferred embodiment of the invention is to use a Black Hole Quencher as quencher, which is energetically adapted to the fluorophore and completely extinguishes the fluorescence of the fluorophore in the bound state.

(35) According to another particularly preferred embodiment of the invention, the fluorescence intensity of the fluorophore reacts insensitively to changes in the pH of the ambient medium.

(36) The use of pH insensitive fluorophores can ensure that pH changes—whether caused by the nature of the sample material or by bacterial metabolites, carbon dioxide content or temperature—do not affect the measurement result. By measuring the fluorescence conditions, an unbiased statement about the enzyme activity in the sample is possible.

(37) In another preferred embodiment of the invention, it is possible to use a self-quenching fluorophore as fluorophore. The self-quenching (self-extinguishing) fluorophore can be, for example, the following fluorescent dyes: Carboxyfluorescein NBD (derivatives of 7-nitrobenz-2-oxa-1,3-diazol-4-yl) BODIPY (derivatives of 4-bora-3a,4a-diaza-s-indacene) DPH (derivatives of 1,6-diphenyl-1,3,5-hexatriene)

(38) The linker L for the covalent linkage of group A (e.g. quencher or fluorophore group) to the β-latcam ring can be or have an ether bond —O— or a secondary amine bond —NH—. Suitable linkers are described as for the carbapenem substrate according to the invention. The linker L must be such that an irreversible coupling of the group A (e.g. quencher or fluorophore or reporter group, depending on what is bound at this point) to the substrate is possible even after its cleavage by carbapenemase and β-lactamase carbapenemase activity. Such a stable linker can be realized by thiol groups, carboimides, succinimides, etc. as described above, in addition to the above-mentioned ether or amine bonds.

(39) The leaving group X for binding to the backbone of the substrate allows a release of the group Z when the substrate is cleaved by the corresponding enzyme. Suitable leaving groups are described above in the context of the carbapenem substrate. In a preferred embodiment of the invention, the leaving group may be or contain a thioether —S— or a thioether compound —S—R— or a sulphonic acid ester (—O—SO2-) or a sulphonic acid ester compound —O—SO2-R). The leaving groups designated X in the structural formulae of the substrates, which after enzymatic cleavage of the β-lactam ring favour the leaving of the group designated Z (e.g. the fluorophore or quencher group), include those which can stabilize the negative charge by resonance. In addition to the thioethers or thioether derivatives mentioned above, sulphonates and thiols, e.g. in the form of thiophenolates, and phenols or substituted phenoxides (R—O—Ar) (with Ar=aryl) are also mentioned here as further examples. The leaving groups can also be carbamates, carbonates, thiocarbamates or thiocarbonates. In addition, the use of inorganic esters (e.g. phosphate derivatives) is also possible.

(40) In accordance with a preferred embodiment of the invention, the change in fluorescence intensity is measured using highly sensitive measuring technology, in particular a photomultiplier. The high sensitivity of the fluorescence measurement made possible by the photomultiplier allows a specific and meaningful real-time detection of target enzymes (carbapenemases, ESBL) with a low detection limit without time-consuming sample preparation with a compact and simple fluorescence measurement arrangement that also includes at least one light source for excitation of the fluorophore. As described above, the detection of an alternatively used reporter group can also be performed within the framework of the detection method described here.

(41) The invention also specifies a method for the phenotypic detection of resistant bacteria, in which the enzymes of a bacterial culture are released by lysis into a lysate and this lysate is then subjected to a method described above as a sample to be examined. In this way, the presence of resistance-conferring carbapenemases that cleave the β-lactam ring can be phenotypically detected. According to the invention, lysis of the bacterial culture can be achieved by adding a lysis buffer and/or a mechanical lysis method, in particular an ultrasound method.

(42) In contrast to PCR-based, molecular-biological detection methods (genotypic detection methods), the inventive method thus examines the actual (phenotypic) characteristics of the bacteria. Thus, the inventive phenotypic detection method is independent of the genetic variability of the MRGN with respect to these characteristics, thus enabling the detection of pathogens that are not detectable by PCR-based methods (e.g. due to genetic instabilities and a large number of different enzyme classes/subtypes for which many different pairs of primers need to be developed and constantly revalidated).

(43) Preferably, the detection method is a method for the detection of multi-resistant gram-positive or gram-negative bacteria, in particular 3MRGN or 4MRGN. In clinical everyday life, these cause increasing problems, especially in the treatment of infections such as pneumonia and urinary tract infections.

(44) In a preferred embodiment of the method described above, the bacterial culture is first placed in a selective detection medium in which all non-resistant bacteria are killed.

(45) Before adding the lysis buffer, it is particularly useful to measure the cell count by measuring the optical density of the sample and/or by dynamically measuring the scattered light of the sample. If the cell count is large enough, the addition of the lysis buffer can be triggered automatically. The optical density (transmission) can be measured in the range around 600 nm. In an automated measurement of dynamic light scattering (DLS measurement), the cell culture is irradiated with laser light and the fluctuations in the intensity of the light scattered by the cells are recorded as a function of time. The intensity of the scattered light is proportional to the product of concentration and molecular mass of the cells.

(46) In this context, a microfluidic system may be used comprising a container containing a lysis buffer solution and a pump to pump the solution from the container into the sample vessel (e.g. a multi-chamber cuvette). In addition, a protease inhibitor (or a mixture of inhibitors of different specificity) can be added to the buffer to delay degradation of the enzymes. As an alternative or in addition, an adjustable ultrasonic homogenizer can be used to introduce ultrasound into the sample for the bacterial lysis.

(47) The invention also provides for the use of one of the inventive methods described above in at least one of the following areas: clinical diagnosis of patient samples, examination of smears taken from equipment surfaces or other surfaces as part of a hygiene control, examination of toxicological or pharmacological substances. In the latter area, a method according to the invention is particularly suitable for the detection of surviving multi-resistant carbapenemase-forming bacteria within the framework of active substance screening.

(48) A method according to the invention for the phenotypic detection of β-lactam ring cleaving enzymes (carbapenemases and ESBL) or for the detection of resistant bacteria, in particular multi-resistant gram-negative bacteria (MRGN), is fluorescence-based and is performed directly from the sample material. As a result, such a method can be used in many different ways. Since there are almost no effective therapeutics against MRGN infections, it will be one of mankind's future tasks to develop new active substances. Here, a method according to the invention can be used to identify new substances that have an antibiotic effect on MRGN, e.g. by searching large substance libraries for active substances.

(49) Finally, the subject matter of the invention is also a device for phenotypically detecting carbapenemase producers, comprising means for adding a substrate having a carbapenem backbone as defined above, a fluorescence excitation source for optically exciting the reaction products, and a measuring device for quantitatively determining the fluorescence intensity of the released fluorophore.

(50) The term “fluorophore” refers to a fluorescent chromophore. When selecting the fluorophore, care is taken to ensure that its fluorescence emission is preferably not dependent on the pH value of the surrounding medium. Since the pH value is subject to a variety of interfering factors (for example as a result of acids produced by bacteria during the metabolism of nutrients), the use of pH-independent fluorophores is preferred. If pH sensitive fluorophores or quenchers are used, it must be ensured that the reaction takes place in a sufficiently buffered solution.

(51) The term “quencher” refers to a molecule or part of a compound capable of reducing the emission of a fluorophore group. Quenching can take place through a variety of mechanisms, including Förster resonance energy transfer, photo-induced electron transfer, paramagnetic enhancement of intersystem crossing, Dexter transfer (exchange interactions), and excitation coupling, such as the formation of dark complex compounds.

(52) The term “β-lactam ring cleaving enzyme” means carbapenemase and β-lactamase, in particular extended spectrum β-lactamase (ESBL). Equivalent, the term “target enzyme” is used here.

(53) The term “solid phase” refers to any solid surface to which the substrates according to the invention can be coupled and which remains inert during the course of the described test procedures. The coupling of the substrate may be covalent or non-covalent, wherein, if a non-covalent coupling to the solid surface is selected, the non-covalent bond is sufficiently stable in the course of the detection procedures to distinguish the enzyme-conferring cleavage of the substrate from a spontaneous separation, preferably the non-covalent coupling of the substrate also remains stable under the test conditions.

(54) The following combinations are to be mentioned as functional fluorophore/quencher pairs for enzyme detection in detail and exemplarily: Fluorophore: Quasar 570-Quencher: Deep Dark Quencher II, Fluorophore: Kal Fluor Red 610-Quencher: BHQ3, Fluorophore: ATTO647N-Quencher: BHQ2, Fluorophore: BODIPY 581/591-Quencher: BHQ2, Fluorophore: Alexa Fluor 430-Quencher: Eclipse Dark Quencher, Fluorophore: Alexa Fluor 555-Quencher: BHQ2, Fluorophore: Cy3-Quencher: BHQ1, Fluorophore: Alexa Fluor 610-Quencher: BHQ, Fluorophore: DY633-Quencher: BHQ3, Fluorophore: ETERNEON YELLOW-Quencher: BHQ1, Fluorophore: ETERNEON RED-Quencher: BHQ3.

(55) In the initial substrate Q-M.sub.1-FI or FI-M.sub.1-Q, the fluorophore and quencher are in close spatial proximity to each other, so that the light energy emitted by the fluorophore is quenched (i.e. reduced) by the quencher. In particular, the quencher absorbs the fluorescence energy of the fluorophore by Förster resonance energy transfer (FRET) and thus prevents emission as fluorescence. The efficiency of FRET depends on the fluorescence quantum yield of the fluorophore, the removal of fluorophore (donor) to quencher (acceptor) and the overlap integral of the fluorescence emission of the fluorophore (donor) and the absorption of the quencher (acceptor). Energy transfer is most efficient when a donor fluorophore with a high fluorescence quantum yield (preferably close to 100%) is paired with a quencher that has a large extinction coefficient at wavelengths coinciding with the emission of the donor.

(56) As a result of the catalytic cleavage of the β-lactam substrate by the respective target enzyme, the connection between the fluorophore or quencher and the catalyzed substrate is also removed. The fluorescence of the non-quenched fluorophore released or remaining in the metabolite can then be detected in the solution as a significant change in the emission of the fluorophore. This requires optical excitation of the sample by excitation radiation from a fluorescence excitation source. In response, the released (fluorescent) fluorophores in the sample emit radiation with a wavelength different from the excitation wavelength. Optical detectors then collect the emission from the sample, whereby the increase in fluorescence intensity can be detected evanescent or by surface screening. If one considers that the measured fluorescence intensity of the sample is proportional to the enzymatic conversion of the substrate, conclusions can be drawn about the enzyme activity or concentration of enzymes cleaving the β-lactam ring via an accompanying calibration series, if a quantitative evaluation is desired.

(57) The term “carbapenemase former” refers to a microorganism capable of forming the enzyme carbapenemase and thereby degrading carbapenem antibiotics. Preferably the microorganism is a bacterium. This bacterium can be gram-negative, or a gram-positive. Examples of gram-negative pathogens are enterobacteria (Escherichia coli, Salmonella, Shigella, Klebsiella, Proteus, Enterobacter) as well as the genera Pseudomonas, Legionella, Neisseria, Rickettsia, Streptobacillus moniliformis, Meningococcus, Chlamydophila, Chlamydia and Spirochaeta Bacteroidetes. Gram-negative bacteria of particular clinical relevance include Pseudomonas aeruginosa, Acinelobacter baumannii, Haemophilus influenzae, Helicobacter pylori, Vibrio cholerae, and Klebsiella penumoniae. Examples of gram-positive bacteria are Actinobacteria, such as those of the genera Actinomyces and Streptomyces, and species of the strain Firmicutes, such as those of the genera Streptococcus, Enterococcus, Staphylococcus, Listeria, Bacillus, Clostridium, and Lactobacillus. Clinically relevant gram-positive bacteria include Staphylococcus aureus, Streptococcus pyogenes, Clostridium difficile, or Listeria monocytogenes.

(58) Examples of 3MRGN and 4MRGN pathogens include K. pneumoniae, E. coli, P. mirabllis, E. cloacae, P. aeruginosa and A. baumannii.

(59) Two preferred embodiments, namely a detection method for extended spectrum β-lactamase (ESBL) according to FIG. 1 and a detection method for carbapenemase covered by the present invention according to FIG. 2, are described in detail below.

(60) FIG. 1 shows the principle of the detection of an extended spectrum β-lactamase as an enzymatic catalyst in abbreviated notation ESBL above the reaction arrow by means of the corresponding reaction equation. β-lactamases are an enzyme class that have been very well investigated due to their clinical relevance (bacterial resistance to β-lactam antibiotics). ESBL, on the other hand, can cleave a larger (expanded) spectrum of β-lactam-containing antibiotics. The ESBL results from a mutation of the well-known β-lactamase. The genes for ESBL are often found on plasmids that can be transferred from bacterium to bacterium.

(61) According to the following structural formula, a substrate labelled with fluorophore and quencher is used as a substrate for the detection of ESBL. Its backbone is formed by a cephalosporin belonging to the class of β-lactam antibiotics.

(62) ##STR00012##

(63) A linker and a leaving group are used to attach the quencher residues and fluorophore residues to the cephalosporin-derived backbone and can facilitate the synthesis of the substrate compound. In the substrate proposed here with the structural formula given above, the fluorophore is connected via a thioether bridge to the 1,3-thiazine six ring of the cephalosporin. The thioether bond is very stable and due to its inductive effect reduces the reactivity of the β-lactam ring towards nucleophiles. The quencher is connected to the -β-lactam ring at the opposite end of the cephalosporin structure via an NH bridge.

(64) Quencher and fluorophore are each molecules selected in such a way that one absorbs light at a wavelength (quencher) at which the other emits (fluorophore). With intact binding to the cephalosporin backbone, the fluorophore (donor) and quencher (acceptor) are closely adjacent, so that energy can be transferred to the quencher by Förster resonance energy transfer, resulting in partial or complete quenching of the donor fluorescence. However, the opening of the β-lactam ring of the cephalosporin by β-lactamase results in a cleavage of the fluorophore from the 3′ position of the cephalosporin. The fluorophore thus becomes free and can diffuse away from the quencher-cephalosporin conjugate. Such a cleavage thus reduces quenching and increases the efficiency of donor fluorescence many times over. The cephalosporin backbone thus serves as a cleavable linker between quencher and fluorophore.

(65) FIG. 2 shows a preferred embodiment of the detection reaction of a carbapenemase according to the invention. The carbapenemase to be detected is again shown in FIG. 2 as an enzymatic catalyst above the reaction arrow. A fluorescence-labelled carbapenem is used as substrate. Carbapenems also belong to the class of β-lactam antibiotics, which have an antibiotic effect based on an irreversible binding to transpeptidases, thereby disrupting the murein synthesis and thus the structure of the cell wall of the bacteria.

(66) However, carbapenems differ from other β-lactams by substituting the sulphur atom in the penam ring with a carbon atom and a double bond in the five-membered ring in position 2-3. They are regarded as reserve antibiotics, which are only used for severe infections with certain bacterial strains that are insensitive to other antibiotics. However, carbapenemase confer resistance to carbapenems, as they very efficiently hydrolyse the amide bond in the β-lactam ring of the carbapenems, whereby carbapenems also lose their effectiveness. At present, however, there is no rapid test method for the detection of carbapenemase producers that allows the detection of this resistance mechanism in clinical sample material or as a culture confirmation test.

(67) The invention provides a remedy by using a chemically modified carbapenem structure with the following structural formula as a substrate for phenotypic fluorescence-based detection of carbapenemase

(68) ##STR00013##

(69) The quencher generally identified with Q is bound e.g. via an ester bond to the hydroxy functionality of the ethyl radical on the beta-lactam backbone. The fluorophore, which is generally identified with FI, is in turn connected to the five membered ring of the carbapenem via a thioether compound (—S—R—) as a linker leaving group, as was the case with the substrate for ESBL detection. The quencher is selected in such a way that it absorbs most of the excitation energy of the fluorophore and thus weakens the self-fluorescence as long as both the quencher and the fluorophore remain bound to the carbapenem backbone.

(70) If, however, in the presence of a carbapenemase in accordance with the detection reaction shown in FIG. 2, the β-lactam ring is cleaved to a quencher-labelled penicillin acid and the fluorophore by hydrolysis at a specific sample temperature adjustable by a temperature control device, then the fluorophore and quencher are spatially separated from each other. With this spatial separation the fluorescence absorption by the quencher is interrupted and the total fluorescence of the reaction increases significantly and reproducibly. Since this increase in fluorescence is proportional to the enzymatic conversion of the carbapenem, the activity or concentration of carbapenemase in the sample can be inferred from a calibration series carried along.

(71) If the presence or activity of at least two target enzymes is to be tested, two or more fluorophores with different emission maxima may be used in parallel to detect the target enzymes in a sample vessel. A simultaneous distinction between different target enzymes is thus possible. The fact that different fluorophores fluoresce simultaneously with different wavelengths (e.g. in the spectral range) when excited together (e.g. laser pulse excitation) is used here. Thus, emission light of a certain wavelength range can be assigned to a specific fluorescence for a specific detection reaction.

(72) The detection reactions described in FIGS. 1 and 2 make it possible for the first time to quickly and reliably detect multi-resistant pathogens such as gram-negative bacteria (MRGN), which form resistance-conferring enzymes that cleave β-lactam rings.

(73) To detect such bacteria, a sample (e.g. a patient sample in the form of blood, sputum, urine, faeces or cerebrospinal fluid or in the form of a smear sample from the oral cavity, nasal cavity, skin or genital tract) is first taken and placed in a liquid selective medium located in a sample vessel (e.g. cuvette) in which all non-resistant bacteria are first killed. For this purpose, the sample vessel is placed in a temperature-controlled measuring device. To release the resistance-conferring enzymes (e.g. If-lactamase and/or carbapenemase), the bacteria are lysed. Lysis is the breaking open of cells, whereby nucleic acids and proteins are released from the inside of the cell. For lysis, the cell material can be treated with a lysis buffer and/or mechanically (e.g. by filling glass beads and placing on a vibrating or shaking apparatus) and/or crushed by targeted ultrasound application. Subsequently, the detection of a target enzyme (or both target enzymes) is realized by using an enzyme-specific reaction with addition of a fluorescence-labelled substrate according to the detection reactions described above and shown in FIGS. 1 and 2. Finally, under optical excitation of the sample by an excitation light source (such as a pulsed LED), the fluorescence of the fluorophores released by the reaction of the target enzymes is measured.

(74) The detection method described above is a phenotypic detection method. In contrast to FOR-based, molecular biological detection (genotypic detection), the actual appearance of the bacteria is investigated. Thus, this method is independent of the genetic variability of MRGN with respect to this appearance and thus also allows the detection of pathogens that cannot be detected by FOR-based methods.

(75) Finally, the invention also comprises a device for the phenotypic detection of carbapenem-resistant pathogens (such as strains of Enterobacteriaceae, Klebsiella pneumoniae) that produce resistance-conferring carbapenemase. Such a device may include an adjustable temperature control means for adjusting the sample temperature, a lysis buffer addition means and/or ultrasonic means for bacteriolysis, a nutrient medium addition means, a substrate addition means, a fluorescence excitation source for optical excitation of the sample and a fluorescence measuring device (e.g. in the form of a highly sensitive photomultiplier) for quantitative detection of the fluorescence intensity of the reaction products. The fluorescence measuring device is preferably coupled with an output device, in particular a display. The device described above can be used in many different ways. In addition to clinical use for the examination of patient samples, smears from the surfaces of devices or other objects can also be analysed as part of a hygiene control. The detection device is therefore also suitable for monitoring decontamination measures.

(76) To measure fluorescence, the sample can be positioned under a photomultiplier and, if necessary, heated by means of a (movable) temperature control device. By heating up, the greatest possible independence from the ambient temperature can be achieved. The optical stimulation for the fluorescence measurements is carried out e.g. by means of light emitting diodes (LEDs), which are mounted above the sample position. A ring-shaped arrangement of the LEDs enables uniform illumination of the surface of the sample carrier. Exchangeable optical filters can be provided between the sample position and the excitation source as well as between the sample position and the photomultiplier, allowing detection of fluorescence in defined bandwidths. The detection of the fluorescence intensities emitted by the reaction products is performed by the photomultiplier, possibly with a filter connected upstream.

(77) Multi-resistant gram-negative pathogens (MRGN) in particular pose new challenges for medicine. Since there are no effective bactericidal therapeutics available for decolonisation of MRGN, doctors and pharmaceutical companies are working intensively on new antibiotics. The device described above can also be used to identify new substances that have an antibiotic effect on MRGN by examining large substance libraries in the form of drug screening. An application of this device is therefore also conceivable in the pharmaceutical industry for the detection of surviving/living MRGN.

(78) In recent years, MRGN has also become increasingly important in the livestock sector, e.g. in fattening plants and in the food industry, due to the improper use of antibiotics and the resulting development of resistance. In addition to the examples given above, the method and device according to the invention is also suitable for the detection of resistant gram-negative bacteria in these areas.

(79) In summary, the presence (e.g. in human serum, pus or urine) of organisms such as bacteria resistant to carbapenem antibiotics may be detected by means of a fluorescence-labelled substrate of the general structural formula (I) or (III) as described herein. Only when active enzymes destroying the β-lactam ring of the carbapenem and other β-lactam antibiotics, such as carbapenemase, occur does the fluorescence change from the intact substrate to one characteristic of the cleavage product. The fluorescence-labelled substrates according to the invention are better than the chromogenic substrates Nitrocephin, CENTA and PADAC of the state of the art, since the substrates according to the invention are stable to the examination material and fluorescence can be detected more sensitively. Furthermore, the measurement of fluorescence of these substrates is insensitive to background signals in self-coloured test materials (e.g. urine or serum).