title: Daptomycin resistance in enterococci is associated with distinct alterations of cell membrane phospholipid content.
abstract: BACKGROUND
  The lipopeptide antibiotic, daptomycin (DAP) interacts with the bacterial cell membrane (CM). Development of DAP resistance during therapy in a clinical strain of Enterococcus faecalis was associated with mutations in genes encoding enzymes involved in cell envelope homeostasis and phospholipid metabolism. Here we characterized changes in CM phospholipid profiles associated with development of DAP resistance in clinical enterococcal strains.
  
  
  METHODS
  Using two clinical strain-pairs of DAP-susceptible and DAP-resistant E. faecalis (S613 vs. R712) and E. faecium (S447 vs. R446) recovered before and after DAP therapy, we compared four distinct CM profiles: phospholipid content, fatty acid composition, membrane fluidity and capacity to be permeabilized and/or depolarized by DAP. Additionally, we characterized the cell envelope of the E. faecium strain-pair by transmission electron microscopy and determined the relative cell surface charge of both strain-pairs.
  
  
  RESULTS
  Both E. faecalis and E. faecium mainly contained four major CM PLs: phosphatidylglycerol (PG), cardiolipin, lysyl-phosphatidylglycerol (L-PG) and glycerolphospho-diglycodiacylglycerol (GP-DGDAG). In addition, E. faecalis CMs (but not E. faecium) also contained: i) phosphatidic acid; and ii) two other unknown species of amino-containing PLs. Development of DAP resistance in both enterococcal species was associated with a significant decrease in CM fluidity and PG content, with a concomitant increase in GP-DGDAG. The strain-pairs did not differ in their outer CM translocation (flipping) of amino-containing PLs. Fatty acid content did not change in the E. faecalis strain-pair, whereas a significant decrease in unsaturated fatty acids was observed in the DAP-resistant E. faecium isolate R446 (vs S447). Resistance to DAP in E. faecium was associated with distinct structural alterations of the cell envelope and cell wall thickening, as well as a decreased ability of DAP to depolarize and permeabilize the CM.
  
  
  CONCLUSIONS
  Distinct alterations in PL content and fatty acid composition are associated with development of enterococcal DAP resistance.
authors.affiliation: Division of Infectious Diseases, Los Angeles Biomedical Research Institute at Harbor-University of California Los Angeles Medical Center, Torrance, California, United States of America.
authors.forename: Nagendra N
authors.initials: NN
authors.lastname: Mishra
authors.forename: Arnold S
authors.initials: AS
authors.lastname: Bayer
authors.forename: Truc T
authors.initials: TT
authors.lastname: Tran
authors.forename: Yousif
authors.initials: Y
authors.lastname: Shamoo
authors.forename: Eugenia
authors.initials: E
authors.lastname: Mileykovskaya
authors.forename: William
authors.initials: W
authors.lastname: Dowhan
authors.forename: Ziqiang
authors.initials: Z
authors.lastname: Guan
authors.forename: Cesar A
authors.initials: CA
authors.lastname: Arias
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects* / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000187:drug effects; D002473:Cell Wall / Q000187:drug effects / Q000378:metabolism; D017576:Daptomycin / Q000494:pharmacology*; D024881:Drug Resistance, Bacterial; D013293:Enterococcus faecalis / Q000166:cytology* / Q000187:drug effects / Q000648:ultrastructure; D016984:Enterococcus faecium / Q000166:cytology* / Q000187:drug effects / Q000648:ultrastructure; D005227:Fatty Acids / Q000378:metabolism; D006801:Humans; D008560:Membrane Fluidity / Q000187:drug effects; D008564:Membrane Potentials / Q000187:drug effects; D010743:Phospholipids / Q000378:metabolism*; D013045:Species Specificity; D017211:Treatment Failure
chemical_list: D000900:Anti-Bacterial Agents; D005227:Fatty Acids; D010743:Phospholipids; D017576:Daptomycin