Mutations in mmpL and in the cell wall stress stimulon contribute to resistance to oxadiazole antibiotics in methicillin-resistant Staphylococcus aureus.

Staphylococcus aureus is a leading cause of hospital- and community-acquired infections, which exhibit broad resistance to various antibiotics. We recently disclosed the discovery of the oxadiazole class of antibiotics, which has in vitro and in vivo activities against methicillin-resistant S. aureus (MRSA). We report herein that MmpL, a putative member of the resistance, nodulation, and cell division (RND) family of proteins, contributes to oxadiazole resistance in the S. aureus strain COL. Through serial passages, we generated two S. aureus COL variants that showed diminished susceptibilities to an oxadiazole antibiotic. The MICs for the oxadiazole against one strain (designated S. aureus COL(I)) increased reproducibly 2-fold (to 4 μg/ml), while against the other strain (S. aureus COL(R)), they increased >4-fold (to >8 μg/ml, the limit of solubility). The COL(R) strain was derived from the COL(I) strain. Whole-genome sequencing revealed 31 mutations in S. aureus COL(R), of which 29 were shared with COL(I). Consistent with our previous finding that oxadiazole antibiotics inhibit cell wall biosynthesis, we found 13 mutations that occurred either in structural genes or in promoters of the genes of the cell wall stress stimulon. Two unique mutations in S. aureus COL(R) were substitutions in two genes that encode the putative thioredoxin (SACOL1794) and MmpL (SACOL2566). A role for mmpL in resistance to oxadiazoles was discerned from gene deletion and complementation experiments. To our knowledge, this is the first report that a cell wall-acting antibiotic selects for mutations in the cell wall stress stimulon and the first to implicate MmpL in resistance to antibiotics in S. aureus.