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Evolution of broad spectrum ?-lactam resistance in an engineered metallo-?-lactamase.

ABSTRACT: The extensive use and misuse of antibiotics during the last seven decades has led to the evolution and global spread of a variety of resistance mechanisms in bacteria. Of high medical importance are ?-lactamases, a group of enzymes inactivating ?-lactam antibiotics. Metallo-?-lactamases (MBLs) are particularly problematic because of their ability to act on virtually all classes of ?-lactam antibiotics. An engineered MBL (evMBL9) characterized by low level activity with several ?-lactam antibiotics was constructed and employed as a parental MBL in an experiment to examine how an enzyme can evolve toward increased activity with a variety of ?-lactam antibiotics. We designed and synthesized a mutant library in which the substrate activity profile was varied by randomizing six active site amino acid residues. The library was expressed in Salmonella typhimurium, clones with increased resistance against seven different ?-lactam antibiotics (penicillin G, ampicillin, cephalothin, cefaclor, cefuroxime, cefoperazone, and cefotaxime) were isolated, and the MBL variants were characterized. For the majority of the mutants, bacterial resistance was significantly increased despite marked reductions in both mRNA and protein levels relative to those of parental evMBL9, indicating that the catalytic activities of these mutant MBLs were highly increased. Multivariate analysis showed that the majority of the mutant enzymes were generalists, conferring increased resistance against most of the examined ?-lactams.

SUBMITTER: Sun S

PROVIDER: S-EPMC3554903 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Evolution of broad spectrum β-lactam resistance in an engineered metallo-β-lactamase.

Sun Song S Zhang Wei W Mannervik Bengt B Andersson Dan I DI

The Journal of biological chemistry 20121203 4

The extensive use and misuse of antibiotics during the last seven decades has led to the evolution and global spread of a variety of resistance mechanisms in bacteria. Of high medical importance are β-lactamases, a group of enzymes inactivating β-lactam antibiotics. Metallo-β-lactamases (MBLs) are particularly problematic because of their ability to act on virtually all classes of β-lactam antibiotics. An engineered MBL (evMBL9) characterized by low level activity with several β-lactam antibioti ...[more]

PMID: 23209299

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Project description:Metallo-β-lactamase (MBL)-producing Enterobacteriaceae are of grave clinical concern, particularly as there are no metallo-β-lactamase inhibitors approved for clinical use. The discovery and development of MBL inhibitors to restore the efficacy of available β-lactams are thus imperative. We investigated a zinc-chelating moiety, 1,4,7-triazacyclononane (TACN), for its inhibitory activity against clinical carbapenem-resistant Enterobacteriaceae MICs, minimum bactericidal concentrations (MBCs), the serum effect, fractional inhibitory concentration indexes, and time-kill kinetics were determined using broth microdilution techniques according to Clinical and Laboratory Standards Institute (CSLI) guidelines. Enzyme kinetic parameters and the cytotoxic effects of TACN were determined using spectrophotometric assays. The interactions of the enzyme-TACN complex were investigated by computational studies. Meropenem regained its activity against carbapenemase-producing Enterobacteriaceae, with the MIC decreasing from between 8 and 64 mg/liter to 0.03 mg/liter in the presence of TACN. The TACN-meropenem combination showed bactericidal effects with an MBC/MIC ratio of ≤4, and synergistic activity was observed. Human serum effects on the MICs were insignificant, and TACN was found to be noncytotoxic at concentrations above the MIC values. Computational studies predicted that TACN inhibits MBLs by targeting their catalytic active-site pockets. This was supported by its inhibition constant (Ki ), which was 0.044 μM, and its inactivation constant (Kinact), which was 0.0406 min-1, demonstrating that TACN inhibits MBLs efficiently and holds promise as a potential inhibitor.IMPORTANCE Carbapenem-resistant Enterobacteriaceae (CRE)-mediated infections remain a significant public health concern and have been reported to be critical in the World Health Organization's priority pathogens list for the research and development of new antibiotics. CRE produce enzymes, such as metallo-β-lactamases (MBLs), which inactivate β-lactam antibiotics. Combination therapies involving a β-lactam antibiotic and a β-lactamase inhibitor remain a major treatment option for infections caused by β-lactamase-producing organisms. Currently, no MBL inhibitor-β-lactam combination therapy is clinically available for MBL-positive bacterial infections. Hence, developing efficient molecules capable of inhibiting these enzymes could be a promising way to overcome this phenomenon. TACN played a significant role in the inhibitory activity of the tested molecules against CREs by potentiating the activity of carbapenem. This study demonstrates that TACN inhibits MBLs efficiently and holds promises as a potential MBL inhibitor to help curb the global health threat posed by MBL-producing CREs.

Dataset Information

Evolution of broad spectrum ?-lactam resistance in an engineered metallo-?-lactamase.

Publications

Evolution of broad spectrum β-lactam resistance in an engineered metallo-β-lactamase.

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