Cervimycin resistant Staphylococcus aureus strains display a vancomycin intermediate resistant phenotype

Resistance to antibiotics is an emerging problem and necessitates novel antibacterial therapies. Cervimycins A‒D are natural products of Streptomyces tendae HKI 0179 with promising activity against multidrug resistant staphylococci and vancomycin resistant enterococci. We studied the mode of action of cervimycin C and D by selection of cervimycin resistant (CmR) Staphylococcus aureus strains. Genome sequencing of CmR mutants revealed amino acid exchanges in the essential histidine kinase WalK, the Clp protease proteolytic subunit ClpP or the Clp ATPase ClpC, and the heat shock protein DnaK. Interestingly, all characterized cervimycin resistant mutants harbored a combination of mutations in walK and clpP or clpC. Mutations in the Clp system abolished ClpP or ClpC activity, and the deletion of clpP rendered S. aureus but not B. subtilis cervimycin resistant. The essential gene walK was the second mutational hotspot in the cervimycin resistant S. aureus mutants, which decreased WalK activity in vitro and generated a vancomycin intermediately resistant phenotype, with a thickened cell wall, a slower growth rate, and reduced cell lysis. Transcriptomic and proteomic analysis revealed massive alterations in the CmR strains , with major alterations in the heat shock regulon, the metal ion homeostasis and the carbohydrate metabolism. Taken together, compensatory mutations in cervimycin resistant mutants induced a VISA  phenotype in S. aureus, suggesting cell wall metabolism or the ClpCP proteolytic system as primary target of the polyketide antibiotic 3 replicates of wildtype S. aureus SG511 Berlin, SG511 derived cervimycin resistant mutants and reverted mutants