The probes were first tested for their antibiotic activity against pathogenic S. aureus (NCTC 8325) and E. coli (MM28, Figure ). As expected, both P2-probes with the derivatized acid moiety, that is essential for antibiotic activity of fluoroquinolones, showed significant (400-to >1600-fold) decreases in their minimal inhibitory concentrations (MICs) both in Grampositive S. aureus and Gram-negative E. coli. Satisfyingly, probes Cipro P1 and Levo P1 both displayed only a 12.5-to 25-fold reduction in the MIC in S. aureus suggesting only slight perturbations in the uptake and/or target engagement. In E. coli, P1-probes exhibited a 100fold potency drop compared to their parent compound, which is most likely due to limited uptake, as the activities in the LPS-deficient E. coli strain RFM795 only decreased 4-fold.