The dramatic rise in prevalence of multi-resistant enterococci in United States hospitals over the past decade has limited therapeutic options, affected morbidity and mortality and increased the cost of caring for seriously ill hospitalized patients. The expression of resistance to vancomycin has received the most attention during this time. However, it is equally problematic that virtually all vancomycin-resistant enterococci (VRE) are Enterococcus faecium that express resistance to high levels of ampicillin. While it is clear that ampicillin resistance in E. faecium requires expression of low affinity penicillin-binding protein 5 (PBP5), the correlation between the amounts of detectable PBP5 and the level of ampicillin resistance is not exact. Several point mutations in pbp5 have been identified in strains expressing high-level ampicillin resistance, but the specific contributions of these mutations to the levels of resistance have never been assessed. We have identified the first transferable ampicillin resistance described from E. faecium in a VRE strain from Northeast Ohio. The pbp5 gene conferring resistance in this isolate possesses several mutations that have been associated with high-level ampicillin resistance in other E. faecium isolates. Curiously, levels of ampicillin resistance expressed by transconjugant E. faecium strains are not equivalent to those expressed by the donor, despite documentation that equivalent amounts of PBP5 are produced. In the past two years, we have acquired evidence that levels of ampicillin resistance expressed correlate with transcription (but not necessarily translation) of an upstream open reading frame designated ftsWEf.
The specific aims of this proposal are to: 1) perform site directed mutagenesis of E. faecium pbp5 to determine the functional (MIC, affinity) and structural importance of specific mutations. With collaborations in France and Switzerland, we now possess the molecular expertise to create the mutants and analyze their functional impact and determine the crystal structure; 2) to investigate the role of the putative upstream repressor psr in regulating expression of ampicillin resistance in E. faecium; 3) to investigate the mechanisms by which transcription of ftsWEf impacts the levels of ampicillin resistance expressed by E. faecium; 4) to assess whether upstream open reading frames designated nanE-Ef and ywrF-Ef affect levels of ampicillin resistance expressed and 5) to determine whether the peptidoglycan precursors differ in sensitive and resistant strains. These investigations will yield new insights into what is arguably the most resistant nosocomial pathogen of our time by providing important structure-function correlations for PBP5, correlations which may be important for the development of newer and better inhibitory compounds. They will also yield important new information on mechanisms of cell wall synthesis in E. faecium and other Gram-positive bacteria as well as on the mechanisms by which ampicillin resistance in E. faecium is regulated.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
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Bacteriology and Mycology Subcommittee 2 (BM)
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Case Western Reserve University
Internal Medicine/Medicine
Schools of Medicine
United States
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García-Solache, Mónica; Rice, Louis B (2016) Draft Genome Sequence of Vancomycin-Susceptible, Ampicillin-Intermediate Enterococcus faecium Strain D344RRF. Genome Announc 4:
Desbonnet, Charlene; Tait-Kamradt, Amelia; Garcia-Solache, Monica et al. (2016) Involvement of the Eukaryote-Like Kinase-Phosphatase System and a Protein That Interacts with Penicillin-Binding Protein 5 in Emergence of Cephalosporin Resistance in Cephalosporin-Sensitive Class A Penicillin-Binding Protein Mutants in Enterococcus faeci MBio 7:e02188-15
García-Solache, Mónica; Rice, Louis B (2016) Genome Sequence of the Multiantibiotic-Resistant Enterococcus faecium Strain C68 and Insights on the pLRM23 Colonization Plasmid. Genome Announc 4:
Triboulet, Sébastien; Bougault, Catherine M; Laguri, Cédric et al. (2015) Acyl acceptor recognition by Enterococcus faecium L,D-transpeptidase Ldtfm. Mol Microbiol 98:90-100
Hugonnet, Jean-Emmanuel; Haddache, Nabila; Veckerlé, Carole et al. (2014) Peptidoglycan cross-linking in glycopeptide-resistant Actinomycetales. Antimicrob Agents Chemother 58:1749-56
Triboulet, Sébastien; Dubée, Vincent; Lecoq, Lauriane et al. (2013) Kinetic features of L,D-transpeptidase inactivation critical for ?-lactam antibacterial activity. PLoS One 8:e67831
Lecoq, Lauriane; Dubée, Vincent; Triboulet, Sébastien et al. (2013) Structure of Enterococcus faeciuml,d-transpeptidase acylated by ertapenem provides insight into the inactivation mechanism. ACS Chem Biol 8:1140-6
Papp-Wallace, Krisztina M; Senkfor, Baui; Gatta, Julian et al. (2012) Early insights into the interactions of different ?-lactam antibiotics and ?-lactamase inhibitors against soluble forms of Acinetobacter baumannii PBP1a and Acinetobacter sp. PBP3. Antimicrob Agents Chemother 56:5687-92
Galloway-Pena, Jessica R; Rice, Louis B; Murray, Barbara E (2011) Analysis of PBP5 of early U.S. isolates of Enterococcus faecium: sequence variation alone does not explain increasing ampicillin resistance over time. Antimicrob Agents Chemother 55:3272-7
Triboulet, Sébastien; Arthur, Michel; Mainardi, Jean-Luc et al. (2011) Inactivation kinetics of a new target of beta-lactam antibiotics. J Biol Chem 286:22777-84

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