CARBAPENEMASE AND ANTIBACTERIAL TREATMENT

Abstract

The present invention relates to a carbapenemase and methods using said carbapenemase such as detection methods, screening methods, predictive methods and therapeutic uses.

Claims

1. A FRI-1 carbapenemase comprising an amino acid sequence defined by SEQ ID NO: 1 or having at least 80% amino acid sequence identity with the amino acid sequence defined by SEQ ID NO: 1.

2. A nucleic acid sequence encoding a carbapenemase comprising an amino acid sequence defined by SEQ ID NO: 1 or having at least 80% amino acid sequence identity with the amino acid sequence defined by SEQ ID NO: 1.

3. The nucleic acid sequence according to claim 2 which is defined by SEQ ID NO: 2.

4. A method for detecting the presence of a FRI-1 carbapenemase comprising an amino acid sequence defined by SEQ ID NO: 1 or having at least 80% amino acid sequence identity with the amino acid sequence defined by SEQ ID NO: 1 in a biological sample, comprising a step of contacting said biological sample with a binding reagent specific for said FRI-1 carbapenemase.

5. An antibody or an aptamer which specifically binds to a FRI-1 carbapenemase comprising an amino acid sequence defined by SEQ ID NO: 1 or having at least 80% amino acid sequence identity with the amino acid sequence defined by SEQ ID NO:1.

6. A kit comprising an antibody or an aptamer which specifically binds to a FRI-1 carbapenemase comprising an amino acid sequence defined by SEQ ID NO: 1 or having at least 80% amino acid sequence identity with the amino acid sequence defined by SEQ ID NO:1; or a probe or a set of primers which specifically hybridizes to a nucleic acid sequence encoding the FRI-1 carbapenemase.

7. A method for detecting the presence of a nucleic acid sequence encoding a carbapenemase comprising the amino acid sequence defined by SEQ ID NO: 1 or having at least 80% amino acid sequence identity with the amino acid sequence defined by SEQ ID NO:1 in a biological sample, comprising a step of contacting said biological sample with a nucleic acid molecule which specifically hybridizes to said nucleic acid sequence.

8-9. (canceled)

10. A probe or a set of primers which specifically hybridizes to a nucleic acid sequence encoding a FRI-1 carbapenemase comprising an amino acid sequence defined by SEQ ID NO: 1 or having at least 80% amino acid sequence identity with the amino acid sequence defined by SEQ ID NO:1.

11. The method according to claim 10, said method comprising the steps of: (i) providing a FRI-1 carbapenemase comprising an amino acid sequence defined by SEQ ID NO: 1 or having at least 80% amino acid sequence identity with the amino acid sequence defined by SEQ ID NO:1 and a substrate of the FRI-1 carbapenemase (ii) bringing the candidate substance to be tested into contact with the FRI-1 carbapenemase and the substrate of step i); (iii) determining the activity of said FRI-1 carbapenemase in presence of the candidate substance to be tested; (iv) comparing the catalytic activity of said carbapenemase determined at step iii) with the catalytic activity of said carbapenemase in the absence of said candidate substance; and (v) positively selecting the candidate substance that inhibits the catalytic activity of said carbapenemase.

12. A method for determining whether a microorganism is resistant to a -lactam compound comprising the step of detecting in said microorganism the presence of a nucleic acid encoding a FRI-1 carbapenemase comprising an amino acid sequence defined by SEQ ID NO: 1 or having at least 80% amino acid sequence identity with the amino acid sequence defined by SEQ ID NO:1, wherein the presence of said nucleic acid is indicative that said microorganism is resistant to -lactams.

13. A method for predicting whether or not a patient that is infected with a microorganism will respond to treatment with a -lactam compound, comprising isolating the microorganism responsible for the infection and determining whether said microorganism is resistant to -lactam compounds by detecting in said microorganism the presence of a nucleic acid encoding a FRI-1 carbapenemase comprising an amino acid sequence defined by SEQ ID NO: 1 or having at least 80% amino acid sequence identity with the amino acid sequence defined by SEQ ID NO:1, wherein the presence of said nucleic acid is indicative that said microorganism is resistant to -lactams, and that said patient will not respond to treatment with a -lactam compound.

Description

FIGURES

[0177] FIG. 1: Antibiotic susceptibility testing to -lactams antibiotics of E. cloacae FRI-1 expressing FRI-1.

[0178] FIG. 2: Antibiotic susceptibility testing to non -lactam antibiotics of E. cloacae FRI-1 expressing FRI-1.

[0179] FIG. 3: Genetic environment of the bla FRI-1 gene in E. cloacae FRI-1.

EXAMPLE

Material and Methods

[0180] Bacterial Strains and Plasmids:

[0181] Identification of Enterobacter cloacae FRI-1 was performed by using the API 20E system (bioMrieux, Marcy l'Etoile, France). E. coli TOP10 was used as host strain for cloning and E. coli J53 (resistant to azide) as host for conjugation assays.

[0182] Antimicrobial Agents and MIC Determinations:

[0183] The antimicrobial agents and their sources have been described elsewhere. Susceptibility testing was performed by disk diffusion assay (Sanofi-Diagnostic Pasteur, Marnes-la-Coquette, France), as previously described. The minimal inhibitory concentrations (MICs) were determined by Etest (AB biodisk; Solna, Sweden) on Mueller-Hinton agar plates at 37 C. Results of susceptibility testing were recorded according to the CLSI guidelines. MBL detection tests were performed using an E-test strip (AB Biodisk).

[0184] Cloning Experiments, PCR and DNA Sequencing:

[0185] PCR screening for class A carbapenemases (bla.sub.KPC, bla.sub.IMI bla.sub.SME), MBL encoding genes bla.sub.VIM, bla.sub.IMP bl, for extended-spectrum -lactamases (ESBLs) encoding genes bla.sub.TEM bla.sub.SHV, and bla.sub.CTX-M, and for plasmid-mediated cephalosporinase gene (bla.sub.CMY bla.sub.ACC), were performed as described. In order to identity the bla FRI-1 gene, blunt clonings were performed using HindIII and Sau3 A restriction enzymes using pBKCMV as a cloning vector and E. coli TOP10 as the recipient strain. In order to express in the bla.sub.FRI-1 gene in an identical background, cloning of this gene was performed into E. coli TOP10 as described, using the PCRBlunt TOPO cloning kit (Invitrogen, Cergy-Pontoise, France) followed by selection on plates containing 50 g/ml of amoxicillin and 30 g/ml of kanamycin. The PCR amplicon encompasses the entire sequence of the bla.sub.FRI-1 gene. Those amplicons did not include the original promoter region of the bla.sub.FRI-1 gene, in order to express those genes under the control of the same promoter provided by plasmid pCR-Blunt-TOPO. Corresponding recombinant strains were used for MIC determinations.

[0186] Both strands of the cloned DNA inserts of recombinant plasmids were sequenced by using an Applied Biosystems sequencer (ABI 377). The nucleotide and deduced protein sequences were analysed with software available over the Internet from the National Center for biotechnology Information website (http://www.ncbi.nlm.nih.gov/BLAST/).

[0187] -Lactamase Purification:

[0188] Cultures of E. coli TOP10 harboring recombinant plasmid pFRI-1 was grown overnight at 37 C. in 2 liters of TS broth containing ticarcilllin (50 g/ml) and ticarcillin (100 g/ml). -Lactamase FRI-1 was purified by ion-exchange chromatography. Briefly, the bacterial suspension was pelleted, resuspended in 50 ml of 20 mM Tris-Ethanolamine buffer (pH 7.2). The pellet was then was sonicated, cleared by ultracentrifugation and treated with DNase. The extract was then dialyzed against 20 mM Bis-Tris-ethanolamine (pH 7.2) and loaded onto a preequilibrated Q-sepharose column on an AKTA purifier (GE Healthcare, USA) followed by a second Q-sepharose column using a Tris-ethaloamine buffer pH9.5. The -lactamase-containing fractions were eluted with a linear NaCl gradient (0 to 1 M). Fractions containing the highest -lactamase activities were pooled and subsequently dialyzed overnight against 20 mM Tris-ethanolamine buffer (pH 7.2). The -lactamase activity was determined qualitatively using nitrocefin. The protein content was measured using the Bio-Rad DC protein assay. The purification factor was measured by comparing the activities of the FRI-1 crude extract and purified enzyme using 100 M imipenem as substrate. The isoelectric point of the FRI-1 enzyme was determined using an electrofocalisation system on polyacrylamide gel (Clean Gel IEF, GE Healthcare, USA) and IEF standards (IEF standards, Bio-Rad).

[0189] Kinetic Studies:

[0190] Kinetic measurements (k.sub.cat and K.sub.m) of purified -lactamase FRI-1 were performed spectrophotometrically as described previously and compared to those previously published for class A carbapenemases.

[0191] Plasmid Content, Conjugation Assays, and Transformation:

[0192] Plasmid DNAs of E. cloacae FRI-1 was extracted by using the Kieser method. E. coli NCTC50192, harboring four plasmids of 154, 66, 48 and 7 kb, was used as the size marker for plasmids. Plasmid DNAs were analyzed by agarose gel electrophoresis as described previously. Direct transfer of the -lactam resistance markers into azide-resistant E. coli J53 was attempted by liquid mating out assays at 37 C. Selection of the transformants was performed on agar plates supplemented with aztreonam (50 g/ml), azide (100 g/ml) and nalidixic acid (20 g/ml). Plasmid encoding the FRI-1 determinant was typed according to known typing scheme.

[0193] Results:

[0194] Characteristics of E. cloacae FRI-1:

[0195] Isolate E. cloacae FRI-1 was resistant or had a decreased susceptibility to all -lactams, including imipenem, meropenem, and ertapenem (FIG. 1). This strain was mostly susceptible to non--lactam antibiotics (FIG. 2).

[0196] Genetic Support of the bla.sub.FRI-1 Gene:

[0197] Plasmid analysis identified two plasmids of ca. 150-160 kb and ca. 60-70 kb, in E. cloacae FRI-1. Conjugation experiments failed to produce E. coli transconjugants exhibiting using E. coli INE-1 s as the donor. This indicates that the FRI-1 plasmid was neither self-conjugative nor mobilizable by one of the natural plasmids contained in E. cloacae FRI-1. A single FRI-1 plasmid was obtained after electrotransformation experiments of ca. 160-170 kb in size. This plasmid was not typeable according to typing plasmid scheme and did not confer any additional resistance markers. Using a sequencing primer walking strategy, the analysis of the genetic environment of the bla.sub.FRI-1 gene in isolate FRI-1 showed that it was bracketed by an ISRaq1 element (FIG. 3).

[0198] Characterization of FRI-1:

[0199] -Lactamase FRI-1 is an Ambler class A -lactamase. However it differs significatively from known carbapenemases. This 293 amino acid protein shared 55, 54, 54, 53, 51% amino-acid identity with NMC-A, IMI-1, Sme-1/Sme-1, SFC-1 and KPC-2. It was therefore distantly related to non-Ambler class A carbapenemases such as NDM-1, IMP-1, VIM-1 and OXA-48. Expression of the bla.sub.FRI-1 gene in E. coli TOP10 conferred as expected reduced susceptibility to all -lactams tested (Table A). The highest MIC values were those observed for ceftazidime and aztreonam. As observed for class A carbapenemases, addition of a fixed concentration of clavulanic acid (4 ug/ml) decreased the MIC values for all antibiotic tested. Similar results were obtained after addition of tazobactam (4 g/ml) which is also a classical -lactamase inhibitor of class A carbapenemase (data not shown).

[0200] FRI-1 was purified to near homogeneity (>90%) as determined by SDS-PAGE analysis, and the purification factor was estimated to be 40-fold. Its molecular weight was ca. 30 kDa and its pI value was 8.66. -Lactamase FRI-1 hydrolyzed all tested -lactams including significantly the carbapenems tested (Table B).

TABLE-US-00003 TABLE A MIC values of several -lactams. E. coli TOP10 E. cloacae cloned FRI-1 FRI-1 gene E. coli TOP10 Imipenem 8 0.75 0.2 Imipenem + Clav 4 0.5 0.2 Ertapenem 24 0.12 0.006 Ertapenem + Clav 8 0.03 0.006 Meropenem 3 0.12 0.02 Meropenem + clav 2 0.03 0.02 Ceftazidime 6 1 0.2 Ceftazidime + clav 3 0.38 0.01 Cefotaxime 1 0.25 0.05 Aztreonam 256 256 0.1 Aztreonam + clav 128 8 0.1 Cefepime 0.38 0.06 0.02 Cefpirome 1.5 0.1 0.05

TABLE-US-00004 TABLE B Kinetic parameters of purified FRI-1 FRI-1 K.sub.m K.sub.cat K.sub.cat/Km -Lactam (M) (sec.sup.1) (M.sup.1/sec.sup.1) Penicillin G 567 1,060 0.21 Amoxicillin >5,000 Ticarcillin 393 120 305 Piperacillin >3,000 Cefalotin >3,000 Cefotaxime >5,000 Cefepime 3,400 28 8 Moxalactam >2,600 Aztreonam >5,000 Imipenem 1,614 1,800 1,100 Ertapenem 98 150 1,500 Meropenem 70 45 650

REFERENCES

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