METHOD AND DEVICE FOR THE DETECTION OF CARBAPENEMASES AND CARBAPENEMASE PRODUCERS

20200248226

16/611422

May 04, 2018

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)-M1-(X)—Z, where M1 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.

1. A method for the phenotypic detection of carbapenemase and carbapenemase producers, the method comprising: contacting a substrate of the general structural formula (I) with a sample suspected of containing the carbapenemases and/or carbapenemase producers: A-(L)-M1-(X)—Z   (I) wherein M1 is a carbapenem backbone of formula (M1): [chemical expression included] wherein R, R1 and R2 are each independently selected from the group consisting of H, a C1-C6 alkyl (methyl, ethyl, propyl, butyl, t butyl, iso propyl optionally substituted with —OH, ═O, —NH2, ═S, or —SH—), optionally substituted oxyalkyl, halogen, —OH, —SH, an optionally substituted alkenyl, an optionally substituted aromatic compound, an optionally substituted heterocyclic compound, a primary or secondary amine, ester, ether, thiol and thioether; wherein the group A-(L)- is linked to the beta-lactam ring of the carbapenem backbone, and wherein the group —(X)—Z is linked to the five-membered ring of the carbapenem backbone, A and Z form a fluorophhore/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 fluorophhore/quencher pair, when A is a quencher, Z is a fluorophore, and when A is a fluorophore, Z is a quencher, L is an optional linker for coupling A to the carbapenem skeleton, and X is an optional leaving group for covalently linking Z to the carbapene backbone, and wherein the group Z is released after enzymatic cleavage of the substrate by 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 CH2, O, S, (—CH═CH)n—CH2— and CO, wherein n is an integer from 1 to 4.

4. The method according to claim 1, wherein the substrate has the structural formula (III): [chemical expression included] wherein R1 is absent or is selected from the group consisting of —CH2—, —O—, —S—, —CO—, CHOH, and (—CH═CH)n—CH2—, wherein n is an integer from 1 to 4.

5. The method according to claim 1, wherein prior to the release of the fluorophore or quencher (Z) the fluorescence of the fluorophore is reduced or quenched by Förster resonance energy transfer to the quencher (A) 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 the fluorescence is measured.

6. 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.

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

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

9. 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.

10. 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.

11. The method according to claim 1, wherein leaving group (X) is or comprises a thioether —S—, or a thioether compound —S—R—, or a sulfonic acid ester (—O—SO2—), or a sulfonic acid ester compound (—O—SO2—R).

12. The method according to claim 4, wherein the sample additionally contains a substrate of the structural formula (IV) [chemical expression included] wherein after enzymatic cleavage of the substrate by beta-lactamase the group Z is released.

13. The method according to claim 12, wherein the substrates (III) and (IV) have distinct reporter groups to phenotypically detect carbapenemase and beta-lactamase producers or the enzymes carbapenemase and beta-lactamase (ESBL) in the sample in parallel and simultaneously.

14. The methodMethod according to claim 12 or 13, wherein the substrates (III) and (IV) have fluorophores with different emission maxima.

15. A method for phenotypically detecting resistant bacteria by releasing the enzymes of a bacterial culture into a lysate during lysis and subsequently subjecting the lysate as a sample to be investigated to a method according to claim 1 in order to phenotypically detect the presence of resistance-conferring carbapenemases and/or beta-lactamases with an extended spectrum, preferably multi-resistant gram-positive or gram-negative bacteria, in particular 3MRGN or 4MRGN, are detected by the method.

16. The method according to claim 15, wherein the bacterial culture is first placed in a selective detection medium in which all non-resistant bacteria are killed.

17. The method according to claim 15, wherein the bacteria are lysed after reaching a sufficient cell number, which is determined by measuring the optical density and/or by a dynamic measurement of the scattered light, preferably a lysis buffer is added to the bacterial culture for lysis and/or a mechanical digestion method, in particular an ultrasound method, is used.

18. (canceled)

19. A device for phenotypically detecting carbapenemase and carbapenemase producers comprising means for contacting a substrate of structural formula (I) or (III) as defined in claim 4 with a sample suspected of containing such carbapenemase producers and means for detecting the cleavage products of the substrate formed by carbapenemase activity.

20. The device according to claim 19, wherein the device for detecting the cleavage products comprises a fluorescence excitation source for optically exciting the fluorophore used and a measuring device for measuring the fluorescence of the fluorophore separated from the quencher.

21. The device according to claim 19 further comprising means for adding a lysis buffer and/or ultrasound means to prepare the sample to be tested by lysis of a bacterial culture.

22. The device according to claim 21, further comprising a tempering device for maintaining the bacterial culture and/or sample at a desired temperature before and/or after addition of the substrate.

23. The device according to claim 19, wherein a photomultiplier coupled to output means is provided for amplifying the measurement signal.

24. A substrate for phenotypic detection of carbapenemases and carbapenemase producers as defined in claim 1.

25. A substrate for phenotypic detection of ESBL as defined in claim 11.

26. A solid phase containing the substrate as claimed in claim 25 bonded to its surface.

12; 12; 12; 01; 07; 07

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