Restoration of daptomycin sensitivity with adjunctive cefazolin is associated with C-terminal MprF mutations in MRSA bacteremia isolates

(MRSA) bacteremia treatment failure. Clinical trials have not yielded a clear approach to MRSA combination therapy. Furthermore, there is not a protocol approved by the Clinical Laboratory Standards Institute (CLSI) for testing most possible antibiotic combinations, and methodology used in basic research laboratories for combination testing presents significant hurdles for implementation in clinical laboratories. In response to these challenges, we developed a high-throughput antibiotic combination testing assay to measure the minimum antibiotic concentration(s) required to reach 99% growth inhibition (IC99) and used it to identify antibiotics that can be paired with DAP to restore the DAP susceptibility of DAP-resistant (DAP-R) MRSA. The IC99 recapitulated time-kill curve results and were consistent with results of single drug MIC measurements performed under CLSI conditions. Using this assay, we found that cefazolin (CFZ) restored DAP susceptibility for DAP-R MRSA with L826F and Q692E mutations in the C-terminal region of the synthase domain of the multiple peptide resistance factor MprF. CFZ also improved VAN susceptibility when used in combination with VAN against these isolates. We established the association between the L826F MprF mutation and CFZ improvement in DAP (and VAN) susceptibility using an MprF deletion strain and transcomplementation with WT MprF compared with L826F MprF. This study highlights the IC99 assay's potential for identifying antibiotic treatment for DAP-R MRSA bacteremia and suggests a probable link between MprF synthase domain mutations and CFZ's ability to improve or restore DAP or VAN susceptibility.