title: Whole-genome analyses of Enterococcus faecium isolates with diverse daptomycin MICs.
abstract: Daptomycin (DAP) is a lipopeptide antibiotic frequently used as a "last-resort" antibiotic against vancomycin-resistant Enterococcus faecium (VRE). However, an important limitation for DAP therapy against VRE is the emergence of resistance during therapy. Mutations in regulatory systems involved in cell envelope homeostasis are postulated to be important mediators of DAP resistance in E. faecium. Thus, in order to gain insights into the genetic bases of DAP resistance in E. faecium, we investigated the presence of changes in 43 predicted proteins previously associated with DAP resistance in enterococci and staphylococci using the genomes of 19 E. faecium with different DAP MICs (range, 3 to 48 μg/ml). Bodipy-DAP (BDP-DAP) binding to the cell membrane assays and time-kill curves (DAP alone and with ampicillin) were performed. Genetic changes involving two major pathways were identified: (i) LiaFSR, a regulatory system associated with the cell envelope stress response, and (ii) YycFGHIJ, a system involved in the regulation of cell wall homeostasis. Thr120 → Ala and Trp73 → Cys substitutions in LiaS and LiaR, respectively, were the most common changes identified. DAP bactericidal activity was abolished in the presence of liaFSR or yycFGHIJ mutations regardless of the DAP MIC and was restored in the presence of ampicillin, but only in representatives of the LiaFSR pathway. Reduced binding of BDP-DAP to the cell surface was the predominant finding correlating with resistance in isolates with DAP MICs above the susceptibility breakpoint. Our findings suggest that genotypic information may be crucial to predict response to DAP plus β-lactam combinations and continue to question the DAP breakpoint of 4 μg/ml.
authors.affiliation: Molecular Genetics and Antimicrobial Resistance Unit, Universidad El Bosque, Bogotá, Colombia.
authors.forename: Lorena
authors.initials: L
authors.lastname: Diaz
authors.affiliation: Department of Internal Medicine, Division of Infectious Diseases, University of Texas Medical School at Houston, Houston, Texas, USA University of Houston College of Pharmacy, Houston, Texas, USA.
authors.forename: Truc T
authors.initials: TT
authors.lastname: Tran
authors.affiliation: Department of Internal Medicine, Division of Infectious Diseases, University of Texas Medical School at Houston, Houston, Texas, USA Department of Medicine, Clínica Alemana de Santiago, Universidad del Desarrollo, Santiago, Chile.
authors.forename: Jose M
authors.initials: JM
authors.lastname: Munita
authors.affiliation: Department of Internal Medicine, Division of Infectious Diseases, University of Texas Medical School at Houston, Houston, Texas, USA.
authors.forename: William R
authors.initials: WR
authors.lastname: Miller
authors.affiliation: Molecular Genetics and Antimicrobial Resistance Unit, Universidad El Bosque, Bogotá, Colombia.
authors.forename: Sandra
authors.initials: S
authors.lastname: Rincon
authors.affiliation: Molecular Genetics and Antimicrobial Resistance Unit, Universidad El Bosque, Bogotá, Colombia.
authors.forename: Lina P
authors.initials: LP
authors.lastname: Carvajal
authors.affiliation: The Genome Institute, Washington University at St. Louis, St. Louis, Missouri, USA.
authors.forename: Aye
authors.initials: A
authors.lastname: Wollam
authors.affiliation: Molecular Genetics and Antimicrobial Resistance Unit, Universidad El Bosque, Bogotá, Colombia Department of Internal Medicine, Division of Infectious Diseases, University of Texas Medical School at Houston, Houston, Texas, USA.
authors.forename: Jinnethe
authors.initials: J
authors.lastname: Reyes
authors.affiliation: Molecular Genetics and Antimicrobial Resistance Unit, Universidad El Bosque, Bogotá, Colombia Department of Internal Medicine, Division of Infectious Diseases, University of Texas Medical School at Houston, Houston, Texas, USA.
authors.forename: Diana
authors.initials: D
authors.lastname: Panesso
authors.affiliation: Molecular Genetics and Antimicrobial Resistance Unit, Universidad El Bosque, Bogotá, Colombia.
authors.forename: Natalia L
authors.initials: NL
authors.lastname: Rojas
authors.affiliation: Department of Biochemistry and Cell Biology, Rice University, Houston, Texas, USA.
authors.forename: Yousif
authors.initials: Y
authors.lastname: Shamoo
authors.affiliation: Department of Internal Medicine, Division of Infectious Diseases, University of Texas Medical School at Houston, Houston, Texas, USA.
authors.forename: Barbara E
authors.initials: BE
authors.lastname: Murray
authors.affiliation: The Genome Institute, Washington University at St. Louis, St. Louis, Missouri, USA.
authors.forename: George M
authors.initials: GM
authors.lastname: Weinstock
authors.affiliation: Molecular Genetics and Antimicrobial Resistance Unit, Universidad El Bosque, Bogotá, Colombia Department of Internal Medicine, Division of Infectious Diseases, University of Texas Medical School at Houston, Houston, Texas, USA cesar.arias@uth.tmc.edu.
authors.forename: Cesar A
authors.initials: CA
authors.lastname: Arias
mesh_terms: D019943:Amino Acid Substitution; D000667:Ampicillin / Q000494:pharmacology; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001896:Boron Compounds; D002462:Cell Membrane / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D002473:Cell Wall / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D017576:Daptomycin / Q000494:pharmacology*; D024901:Drug Resistance, Multiple, Bacterial / Q000235:genetics; D016984:Enterococcus faecium / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes; D015870:Gene Expression; D005809:Genes, Regulator; D016680:Genome, Bacterial; D008826:Microbial Sensitivity Tests; D014640:Vancomycin / Q000494:pharmacology; D020713:Vancomycin Resistance / Q000235:genetics
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D001896:Boron Compounds; D005456:Fluorescent Dyes; D014640:Vancomycin; D000667:Ampicillin; D017576:Daptomycin