Characterization of compensatory mutations associated with restoration of daptomycin-susceptibility in daptomycin non-susceptible methicillin-resistant Staphylococcus aureus and the role mprF mutations

The objective of this study was to investigate the underlying mechanism explaining reversion of clinical DAP non-susceptible (NS) MRSA isolates to DAP-susceptible (S) by analysis of genomic and cell wall characteristics of clinical DAP-NS MRSA and DAP-S MRSA isolates as well as in vitro revertant DAP-S MRSA using whole genome sequencing (WGS) and analysis of biological properties. WGS of the 4 clinical DAP-NS MRSA revealed mprF mutations resulting in amino acid substitutions or deletion. These same amino acid substitutions and deletion were also observed in the 4 in vitro revertant DAP-S strains. While WGS identified the presence of the same mprF mutations in both the DAP-NS and in vitro DAP-S revertant strains, new mutations were also detected in other genes and intergenic regions of in vitro DAP-S revertant strains. Transmission electron microscopy to assess cell-wall (CW) thickness of 4 sets strains (pre- and post-DAP therapy isolates and in vitro DAP-S revertant) showed that 3 of the 4 isolates developed increased thickness of the CW after DAP therapy. After reversion to DAP susceptibility, CW thickness was decreased to the same level as DAP-S MRSA. Our results indicate that in vitro conversion of DAP-NS MRSA to DAP-S is independent of mprF gene mutations and may be partially explained by a change in CW thickness. However, as some strains showed no change in the CW, further studies are required to elucidate the different mechanisms of resistance to DAP, and factors for conversion of DAP-NS to DAP-S.