Novel determinant of antibiotic resistance: a clinically selectedStaphylococcus aureus clpPmutant survives daptomycin treatment by reducing binding of the antibiotic and adapting a rod-shaped morphology
Authors: Xu Lijuan , Henriksen Camilla , Mebus Viktor , Guérillot Romain , Petersen Andreas , Jacques Nicolas , Jiang Jhih-Hang , Derks Rico J. E. , Sánchez-López Elena , Giera Martin , Leeten Kirsten , Stinear Timothy P. , Oury Cécile , Howden Benjamin P. , Peleg Anton Y. , Frees Dorte 
Published: March 2023

Daptomycin is a last-resort antibiotic used for treatment of infections caused by Gram-positive antibiotic-resistant bacteria such as methicillin-resistantStaphylococcus aureus(MRSA). Treatment failure is commonly linked to accumulation of point mutations, however, the contribution of single mutations to resistance and the mechanisms underlying resistance remain incompletely understood. Here we show that a single nucleotide polymorphism (SNP) selected during daptomycin therapy inactivates the highly conserved ClpP protease and is causing reduced susceptibility of MRSA to daptomycin, vancomycin, and β-lactam antibiotics as well as decreased expression of virulence factors. Super-resolution microscopy demonstrated that the improved survival of theclpPmutant strain during daptomycin treatment was associated with reduced binding of daptomycin to the septal site and diminished membrane damage. In both the parental strain and theclpPstrain, daptomycin inhibited the inward progression of septum synthesis eventually leading to lysis and death of the parental strain while survivingclpPcells were able to continue synthesis of the peripheral cell wall in the presence of 10 × MIC daptomycin resulting in a rod-shaped morphology. To our knowledge, this is the first demonstration that synthesis of the outer cell wall continues in the presence of daptomycin. Collectively, our data provide novel insight into the mechanisms behind bacterial killing and resistance to this important antibiotic. Also, the study emphasizes that treatment with last-line antibiotics is selective for mutations that, like the SNP inclpP, favor antibiotic resistance over virulence gene expression.

IMPORTANCE

The bacteriumStaphylococcus aureusis a leading cause of life-threatening infections and treatment is challenged by the worldwide dissemination of methicillin-resistantStaphylococcus aureus(MRSA) that are multi-drug resistant. Daptomycin, a cell membrane-targeting cationic lipopeptide, is one of the few antibiotics with activity against MRSA, however, the killing mechanism of daptomycin and the mechanisms leading to resistance are not fully understood. Here we show than an MRSA strain, isolated from the blood of a patient treated with daptomycin, has acquired a mutation that inactivates the ClpXP protease resulting in increased resistance to several antibiotics and diminished expression of virulence genes. Super resolution microscopy showed that the mutant avoids daptomycin-elicited killing by preventing the binding of the antibiotic to the septal site and by growing into a rod-shaped morphology. In summary, this study discloses new perspectives on the mechanism of killing and the mechanism of resistance to an antibiotic of last resort.