Prolonged Exposure to β-Lactam Antibiotics Reestablishes Susceptibility of Daptomycin-Nonsusceptible Staphylococcus aureus to Daptomycin.

Daptomycin-nonsusceptible (DAP-NS)often exhibits gain-in-function mutations in thegene (involved in positive surface charge maintenance). Standard β-lactams, although relatively inactive against methicillin-resistant(MRSA), may prevent the emergence ofmutations and DAP-NS. We determined if β-lactams might also impact DAP-NS isolates already possessing anmutation to revert them to DAP-susceptible (DAP-S) phenotypes and, if so, whether this is associated with specific penicillin-binding protein (PBP) targeting. This study included 25 DAP-S/DAP-NS isogenic, clinically derived MRSA bloodstream isolates. MICs were performed for DAP, nafcillin (NAF; PBP-promiscuous), cloxacillin (LOX; PBP-1), ceftriaxone (CRO; PBP-2), and cefoxitin (FOX; PBP-4). Three DAP-NS isolates were selected for a 28-day serial passage in subinhibitory β-lactams. DAP MICs and time-kill assays, host defense peptide (LL-37) susceptibilities, and whole-genome sequencing were performed to associate genetic changes with key phenotypic profiles. Pronounced decreases in baseline MICs were observed for NAF and LOX (but not for CRO or FOX) among DAP-NS versus DAP-S isolates ("seesaw" effect). Prolonged (28-d) β-lactam passage of three DAP-NS isolates significantly reduced DAP MICs. LOX was most impactful (∼16-fold decrease in DAP MIC; 2 to 0.125 mg/liter). In these DAP-NS isolates with preexistingpolymorphisms, accumulation of additionalmutations occurred with prolonged LOX exposures. This was associated with enhanced LL-37 killing activity and reduced surface charge (both-dependent phenotypes). β-lactams that either promiscuously or specifically target PBP-1 have significant DAP "resensitizing" effects against DAP-NSstrains. This may relate to the acquisition of multiplesingle nucleotide polymorphism (SNPs), which, in turn, affect cell envelope function and metabolism.