Session: Slide Session: Old and New Glyco- and Glyco-Lipo Peptides
Tuesday, October 28, 2008: 12:00 AM
Room: Room 147A
Background: Daptomycin (DAP) is a novel lipopeptide antibiotic with potent activity against Gram positive bacteria approved for treatment of complicated skin and skin structure infections and bacteremia and right-sided endocarditis caused by MSSA and MRSA. Emergence of resistance to DAP is extremely rare, both in the laboratory and in the clinic. Mutations in mprF, responsible for converting phosphatidylglycerol (PG) to lysylphosphatidylglcerol (LPG), have been identified in strains with decreased susceptibility. We developed an LC-MS/MS method to identify and quantify PG and LPG levels, and used this method to characterize the membrane lipid changes associated with mprF mutations in DAP resistant isolates. Methods: Lipids were obtained from exponentially grown cultures by chloroform-methanol extraction and analyzed by LC-MS. Greater than 30 isotopic lipid species were identified for both PG and LPG in a wild type strain. Several of these masses were chosen as compounds of interest and fragmented through Collision Induced Dissociation for identification by accurate mass assignment and isotopic enhanced resolution. Neutral loss and multiple reaction monitoring methods were then used to track individual PG and LPG species. Quantitation was achieved by inclusion of internal deuterium-labeled standards and normalization for phosphorus content. Results: Seven pairs of WT/DapR isolates were examined. PG/LPG ratios decreased in four of the mutants, while in the remaining pairs, no change was seen. No significant changes were observed in the distribution of tail lengths associated with PG and LPG headgroups in the DapR isolates. Conclusions: Increases in LPG content in DapR isolates are consistent with genetic data suggesting gain of function mutations are associated with reduced DAP susceptibility. However, these changes cannot explain the role of mutations in mprF in all DapR isolates.