Daptomycin resistance in methicillin-resistant Staphylococcus aureus is conferred by IS256 insertion in the promoter of mprF along with mutations in mprF and walK

Abstract The mechanisms underlying the emergence of daptomycin-resistant Staphylococcus aureus remain unclear. S. aureus strain 3d0, which was isolated from a patient with bloodstream infections and belongs to the predominant Chinese hospital-associated methicillin-resistant S. aureus (MRSA) clone ST239, was serially passaged on gradient broth containing daptomycin for 34 days. The whole genomes of 3d0 and its serial passage strains were sequenced and compared. Five single nucleotide polymorphisms, four IS256 insertions, and one 39-bp insert occurred in the progress of daptomycin resistance acquisition. IS256 insertion in the mprF promoter region resulted in mprF overexpression. Two novel point mutations in mprF and walK, leading to amino acid substitutions in MprF (G299V and L473I) and WalK (L7Q and Y225N), were proved to increase the MIC of daptomycin by 2–4 times, by allelic replacement experiments. Allelic replacements of both mprF and walK in 3d0 increased the MIC of daptomycin by 4–8-fold, indicating mprF and walK mutations synergistically contribute to daptomycin non-susceptibility. Notably, these mutants acquired resistance without losing fitness, and exhibited decreased expression of cell-wall degradation-related genes. In conclusion, our study revealed novel mutations of MRSA daptomycin resistance acquisition in vitro and several novel mutations in walK and mprF, and it included the first in-depth analysis of the mprF promoter. Our study shed light on how MRSA may acquire daptomycin resistance during daptomycin treatments