Membrane-Targeting Triphenylphosphonium Functionalized Ciprofloxacin for Methicillin-Resistant Staphylococcus aureus (MRSA)

Abstract

Multidrug-resistant (MDR) bacteria have become a severe problem for public health. Developing new antibiotics for MDR bacteria is difficult, from inception to the clinically approved stage. Here, we have used a new approach, modification of an antibiotic, ciprofloxacin (CFX), with triphenylphosphonium (TPP, PPh₃) moiety via ester- (CFX-ester-PPh₃) and amide-coupling (CFX-amide-PPh₃) to target bacterial membranes. In this study, we have evaluated the antibacterial activities of CFX and its derivatives against 16 species of bacteria, including MDR bacteria, using minimum inhibitory concentration (MIC) assay, morphological monitoring, and expression of resistance-related genes. TPP-conjugated CFX, CFX-ester-PPh₃, and CFX-amide-PPh₃ showed significantly improved antibacterial activity against Gram-positive bacteria, Staphylococcus aureus, including MDR S. aureus (methicillin-resistant S. aureus (MRSA)) strains. The MRSA ST5 5016 strain showed high antibacterial activity, with MIC values of 11.12 µg/mL for CFX-ester-PPh₃ and 2.78 µg/mL for CFX-amide-PPh₃. The CFX derivatives inhibited biofilm formation in MRSA by more than 74.9% of CFX-amide-PPh₃. In the sub-MIC, CFX derivatives induced significant morphological changes in MRSA, including irregular deformation and membrane disruption, accompanied by a decrease in the level of resistance-related gene expression. With these promising results, this method is very likely to combat MDR bacteria through a simple TPP moiety modification of known antibiotics, which can be readily prepared at clinical sites.

Keywords: antibiotic conjugates; ciprofloxacin; multidrug resistance bacteria; triphenyl-phosphonium.

Figure 1

Schematic summary of the approach used and the motivation for this work. (a) Applications of triphenylphosphonium (TPP⁺, PPh₃). In this work: X = antibiotic (ciprofloxacin) used to enhance the effect of antibiotics against multidrug-resistant (MDR) bacteria. (b) A schematic diagram showing the mechanism of drug-resistance in bacteria and the mode of action towards ciprofloxacin (CFX) and the functioning mechanism of CFX-PPh₃ derivatives. (c) Chemical structure of CFX and its derivatives; CFX-ester-PPh₃, CFX-amide-PPh₃.

Figure 2

Minimum inhibitory concentration (MIC) and biofilm formation assay for CFX, CFX-ester-PPh₃, and CFX-amide-PPh₃ against methicillin-susceptible S. aureus (MSSA) and MDR Staphylococcus aureus strains. (a) MIC values for different compounds with MSSA and five types of MDR S. aureus. CFX, CFX-ester-PPh₃ and CFX-amide-PPh₃ were serially diluted two-fold in a 96-well microplate according to the concentration range: CFX at 512–0.00005 µg/mL; CFX-ester-PPh₃ and CFX-amide-PPh₃ at 178.5–1.39 µg/mL. (b) Zeta potential of MSSA and methicillin-resistant S. aureus (MRSA) strains. Zeta-potential was measured at 25 °C and determined using phase analysis of scattered light by colloidal particles suspended in de-ionized water (DI H₂O). (c–f) Inhibitory activity against biofilm formation depending on concentration of CFX, CFX-ester-PPh₃, and CFX-amide-PPh₃. Biofilm formed by MSSA and MRSA strains was stained with crystal violet for 10 min and eluted in 33% acetic acid. The biofilm mass was measured at 600 nm of optical density (OD) values. The yellow triangle indicates maximum inhibition of biofilm formation with CFX-amide-PPh₃ against the MRSA strains. All compound treated bacteria were statistically calculated compared to the compound untreated group (as a control). The data for the inhibition of biofilm formation in Gram-negative bacteria are represented in Supporting Information (Figure S6). All experiments were repeated three times. The results are shown as the means ± standard deviation of triplicate independent experiments (* p < 0.05, ** p < 0.001, *** p < 0.0001).

Figure 3

Time-and concentration-dependent efficacy assay for MRSA and MSSA with CFX and CFX-PPh₃ derivatives. (a,b) Time-kill assay of CFX and CFX-PPh₃ derivatives against (a) MSSA and (b) MRSA. Inset A–C representative concentrations; A: 0.5× MIC, B: 1.0× MIC, C: 2.0× MIC. These data are represented as the means ± standard deviation of three results. (c) The growth curve of MSSA and MRSA treated with CFX and CFX-PPh₃ derivatives at 1.0× MIC of each compound. Each point represents the OD values at 600 nm. All experiments were performed in three independent replicates. The 0.5× MIC, 1.0× MIC, and 2.0× MIC values in this experiment are indicated in Table 2.

Figure 4

(a) Transmission electron microscopy (TEM) images of MRSA ST5 5016 cells treated with each compound at 0.5× MIC for 6 h at different magnifications. A: untreated control of MRSA ST5 5016, B: MRSA treated with 0.5× MIC of CFX, C: MRSA treated with 0.5× MIC of CFX-ester-PPh₃, and D: MRSA treated with 0.5× MIC of CFX-amide-PPh₃. The images on the left of all micrographs indicate low-magnification TEM images (low: 40,000× and scale bar: 200 nm) and on the right, high-magnification images of the indicated area (high: 100,000× and scale bar: 100 nm). Each arrow represents the following; the red arrow: intact septum, the black arrow: thinned and irregular cell wall and distortion of the cell, the green arrow: a thorn-like layer present around the outer wall, the purple arrow: partial disintegration of the cytoplasmic membrane, the yellow arrow: loss of cell contents, and the blue arrow: lysis of intracellular contents. (b) Effects of CFX and CFX-PPh₃ derivatives on the relative mRNA expression level of efflux pump genes, such as norA, sepA, and mdeA, in MSSA and MRSA. The gene expression level was measured after CFX and CFX-PPh₃ derivative treatment at 0.5× MIC in comparison to the drug-free growth used as control. Each cycle threshold (Ct) value was normalized to 16S rRNA as internal control, and the normalized fold change was calculated using the delta-delta Ct method, with a drug-free group being the control. Gray bar (MSSA) was assigned a value of 1 and represented the control. The results are shown as the means ± standard deviation of triplicate independent experiments. * Significant, p < 0.05, *** highly significant, p < 0.0001.