Whole Genome Sequencing of Bacterial strains

The increasing infection rate with multidrug resistant bacteria urges the need for novel treatment options. In addition to its potent antiplatelet properties, ticagrelor was found to have antibacterial properties against Gram-positive bacteria, including resistant strains. These findings were corroborated by several studies in cardiovascular patients who had a lower risk of Staphylococcus aureus bacteraemia when receiving ticagrelor instead of other similar antiplatelet agents, clopidogrel or prasugrel. We investigated the mechanism of action of ticagrelor on Gram-positive bacteria. An antibiotic stress triggered bioreporter assay in Bacillus subtilis showed that ticagrelor induced pathways essential for maintaining the integrity of bacterial cell envelope. Ticagrelor caused bacterial membrane depolarization in Bacillus subtilis and methicillin-resistant Staphylococcus aureus (MRSA), and it increased bacteria hydrophobicity, at concentrations that did not inhibit bacterial growth. At bactericidal concentration corresponding to the MIC, ticagrelor disrupted bacterial cytoplasmic membrane as shown by the incorporation of membrane impermeable dye, and the formation of lipid aggregates. Whole genome sequencing of in vitro-generated ticagrelor resistant MRSA clones demonstrated mutations in clpP, clpX and yjbH genes encoding components of the ClpXP protease system. Lipidomic analysis of these ticagrelor resistant MRSA clones revealed several changes in lipid content, with consistently lower levels of cardiolipins (CL), diacylglycerols (DG), and 14:0 fatty acyl chain-bearing phosphatidylglycerols (PG). The addition of exogeneous CL, DG, or PG lipids to MRSA cultures quenched the inhibition of bacterial growth by ticagrelor. Importantly, frequently occurring daptomycin-resistant mutants as well as vancomycin-intermediate S. aureus (VISA) remained susceptible to ticagrelor. No cross-resistance was observed between ticagrelor, daptomycin, or vancomycin. Our study demonstrates that ticagrelor has multiple targets in bacterial cell envelope, thereby retaining activity against multidrug resistant S. aureus.