Abstract
BackgroundThe 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.MethodologyUsing 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.Principal FindingsBoth 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.ConclusionDistinct alterations in PL content and fatty acid composition are associated with development of enterococcal DAP resistance.
Highlights
Enterococci are leading causes of nosocomial infections in the US [1], causing a variety of life-threatening syndromes such as bacteremic infections, urosepsis and meningitis, among others
Distinct alterations in PL content and fatty acid composition are associated with development of enterococcal DAP resistance
Lysylphosphatidylglycerol (L-PG) was the only amino-containing PL detected in E. faecium (Table 1), whereas E. faecalis cell membrane (CM) contained L-PG plus two additional aminocontaining PLs of unknown identity (Table 1, Figure 1)
Summary
Enterococci are leading causes of nosocomial infections in the US [1], causing a variety of life-threatening syndromes such as bacteremic infections (including endocarditis), urosepsis and meningitis, among others. Two species are responsible for the vast majority of enterococcal infections, E. faecalis and E. faecium. The treatment of such infections is often impacted by the increased prevalence of multidrug resistance in these isolates. Ampicillin and vancomycin resistance is present in more than 80% of E. faecium isolates [1], making these compounds almost obsolete for the treatment of this pathogen. 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. We characterized changes in CM phospholipid profiles associated with development of DAP resistance in clinical enterococcal strains
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