β-lactam-induced cell envelope adaptations, not solely enhanced daptomycin binding, underlies daptomycin-β-lactam synergy in methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is a serious clinical threat due to innate virulence properties, high infection rates and the ability to develop resistance to multiple antibiotics, including the lipopeptide, daptomycin (DAP). Acquisition of DAP resistance (DAP-R) in MRSA has been linked with several characteristic alterations in the cell envelope. Clinical treatment of DAP-R MRSA infections has generally involved DAP plus β-lactam combinations, although definable synergy of such combinations varies in a strain-dependent, as well as a β-lactam-dependent manner. We investigated distinct β-lactam-induced cell envelope adaptations of nine clinically-derived DAP-susceptible (DAP-S)/DAP-R strain-pairs following in vitro exposure to a panel of six standard β-lactams (nafcillin, meropenem, cloxacillin, ceftriaxone, cefaclor, or cefoxitin) which differ in their PBP-targeting profiles. In general, in both DAP-S and DAP-R strains, exposure to these β-lactams led to: i) decreased positive surface charge; ii) decreased CM fluidity; iii) increased content and delocalization of anionic phospholipids (i.e., cardiolipin), with the delocalization more pronounced in DAP-R strains; and iv) increased DAP binding in DAP-S (but not DAP-R) strains. Collectively, these results suggest β-lactam-induced alterations in at least three major cell envelope phenotypes (surface charge, membrane fluidity and cardiolipin content) could underlie improved DAP activity, not mediated solely by an increase in DAP binding. This work was presented in part at the 30th European Congress of Clinical Microbiology and Infectious Diseases (ECCMID) Meeting, April 18-21, 2020; Poster #P3403 Note - For ease of presentation, we will utilize the terminology, "DAP-R", instead of "DAP-nonsusceptibility".