Fluoroquinolone structure and translocation flux across bacterial membrane

Abstract

Bacterial multidrug resistance is a worrying health issue. In Gram-negative antibacterial research, the challenge is to define the antibiotic permeation across the membranes. Passing through the membrane barrier to reach the inhibitory concentration inside the bacterium is a pivotal step for antibacterial molecules. A spectrofluorimetric methodology has been developed to detect fluoroquinolones in bacterial population and inside individual Gram-negative bacterial cells. In this work, we studied the antibiotic accumulation in cells expressing various levels of efflux pumps. The assays allow us to determine the intracellular concentration of the fluoroquinolones to study the relationships between the level of efflux activity and the antibiotic accumulation, and finally to evaluate the impact of fluoroquinolone structures in this process. This represents the first protocol to identify some structural parameters involved in antibiotic translocation and accumulation, and to illustrate the recently proposed "Structure Intracellular Concentration Activity Relationship" (SICAR) concept.

Figure 1

Chemical structure and antibacterial activities of fluoroquinolones used. SANO1 structure has been previously described.

Figure 2

Intracellular concentration of Fleroxacin (FLE), Ciprofloxacin (CIP) and a benzo[b]napthyridone (SANO1) obtained with normalization by tryptophan. The accumulation was performed in AG100 (WT), AG100A (AcrAB−) and AG102 (AcrAB++) E. coli strains incubated 5, 15 and 30 min with 2 mg/L of molecules, in the absence or in the presence of CCCP (10 µM). The cells were lysated by HCl-glycine and the fluorescence signal (RFU) was plotted.

Figure 3

Microspectrofluorimetry on bacterial strains (30–40 bacterial cells) during time-course incubation. (A) Kinetics of FLE accumulation (2 mg/L) were followed in AG100, AG100A, AG102 isolated cell with a 2 min sampling time. (B) Kinetics of SANO1 accumulation in individual bacterial cells with 2 mg/L.

Figure 4

Microspectrofluorimetry on individual bacterial strains incubated with FLE/CIP/SANO1 at 2 mg/L calculated from curves as presented in Fig. 3B for SANO1.