871. A Study of Genetic and Functional Alterations in Enterococcus Faecium Resistant to Daptomycin
Session: Oral Abstract Session: Antimicrobial Resistance and Susceptibility
Saturday, October 22, 2011: 10:30 AM
Room: 151AB
Background: Non-susceptibility to daptomycin (DAP) has been mainly reported in S. aureus. We and others have previously found mutations in phospholipid biosynthesis genes, associated with development of S. aureus resistance to DAP. Limited data exist on the genetic alterations and mechanisms that underlie loss of susceptibility to DAP by Enterococci.

Methods: We generated the DAP-resistant Enterococcus faecium GE-1D32 strain (MIC=64μg/ml), after serial passage of the strain GE-1 (MIC=2μg/ml) through increasing DAP concentrations. Using PCR and direct sequencing, we compared the sequences of GE-1 and GE-1D32 strains for three genes homolog to S. aureus genes that were previously found to harbor mutations in DAP-resistant strains: E. faecium Lysyl-tRNA synthetase (ltrs) gene (homolog to the S. aureus mprf), Cardiolipin synthetase (cls) gene (homolog to the S. aureus cls), and CDP-alcohol phosphatidyltransferase (cap) gene (homolog to the S. aureus pgs). In addition, the net cell surface charge of the GE-1 and GE-1D32 strains was compared, in three independent experiments, by quantifying spectrophotometrically (at 410 nm) the binding of the highly cationic molecule cytochrome c (Sigma) to the enterococcal surface, following 15-min incubation.

Results: No alterations were found in E. faecium ltrs, cls or cap genes between GE-1 and GE-1D32. The encoded Lysyl-tRNA synthetase protein was identical to those published in Pubmed (refs ZP_05922238.1, ZP_06445518.1) but differed in three amino acids from sequences of other DAP-susceptible strains (ref ZP_00604896.1). However, the percentage of the positively charged cytochrome c bound by the surface of the cells of DAP-resistant GE-1D32 strain (average 6.4%) was significantly lower than that bound by GE-1 (39.4%; p<0.05). This suggests for increased repulsion of cationic peptides (like DAP) possibly resulting from increased net positive surface charge in resistant strains.

Conclusion: Similar to what has been recently reported for Enterococcus faecalis, the development of DAP-resistance in E. faecium appears genetically distinct from S. aureus, but likely involves mechanisms resulting into cell membrane alterations that affect net surface charge, preventing binding of cationic substances.


Subject Category: A. Antimicrobial agents and Resistance

Spiros Miyakis, Oriol Gasch, Joanna Dakos, Robert Moellering, Jr Jr., MD, FIDSA and George Eliopoulos, MD, FIDSA, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA

Disclosures:

S. Miyakis, None

O. Gasch, None

J. Dakos, None

R. Moellering, Jr Jr., Cubist Pharmaceuticals: Consultant, Consulting fee

G. Eliopoulos, Antimicrobial Therapy Inc: Editor, Licensing agreement or royalty
Theravance Inc: Research Contractor, Research support

Findings in the abstracts are embargoed until 12:01 a.m. EST Thursday, Oct. 20 with the exception of research findings presented at IDSA press conferences.