Anti Gram-Positive Bacteria Activity of Synthetic Quaternary Ammonium Lipid and Its Precursor Phosphonium Salt

Abstract

Organic ammonium and phosphonium salts exert excellent antimicrobial effects by interacting lethally with bacterial membranes. Particularly, quaternary ammonium lipids have demonstrated efficiency both as gene vectors and antibacterial agents. Here, aiming at finding new antibacterial devices belonging to both classes, we prepared a water-soluble quaternary ammonium lipid (6) and a phosphonium salt (1) by designing a synthetic path where 1 would be an intermediate to achieve 6. All synthesized compounds were characterized by Fourier-transform infrared spectroscopy and Nuclear Magnetic Resonance. Additionally, potentiometric titrations of NH₃⁺ groups 1 and 6 were performed to further confirm their structure by determining their experimental molecular weight. The antibacterial activities of 1 and 6 were assessed first against a selection of multi-drug-resistant clinical isolates of both Gram-positive and Gram-negative species, observing remarkable antibacterial activity of both compounds against Gram-positive isolates of Enterococcus and Staphylococcus genus. Further investigations on a wider variety of strains of these species confirmed the remarkable antibacterial effects of 1 and 6 (MICs = 4-16 and 4-64 µg/mL, respectively), while 24 h-time-killing experiments carried out with 1 on different S. aureus isolates evidenced a bacteriostatic behavior. Moreover, both compounds 1 and 6, at the lower MIC concentration, did not show significant cytotoxic effects when exposed to HepG2 human hepatic cell lines, paving the way for their potential clinical application.

Keywords: Gram-positive and Gram-negative bacteria; MICs determination; cytotoxicity studies; membrane permeabilization; multi-drug-resistant bacteria; phosphonium salt (1); quaternary ammonium lipid (6); time-killing experiments.

Figure 1

Chemical structure of QPS 1 (a) and QAS 6 (b) prepared and evaluated as new possible antibacterial agents in this study.

Scheme 1

Synthetic route to achieve the desired QPS and QAS compounds 1 and 6.

Figure 2

FTIR spectrum (KBr) of 1.

Figure 3

¹H NMR spectrum (CDCl₃, 300 MHz) of 1.

Figure 4

¹³C-NMR spectrum (a) and 135 DEPT (b) (CDCl₃, 75.5 MHz) of 1. Red and blue boxes indicate two different regions of spectrum and the related magnifications.

Figure 5

¹H NMR spectrum (CDCl₃, 300 MHz) of 1: magnification of the signal used to calculate the Z/E ratio.

Figure 6

FTIR spectrum (KBr disc) of Z/E 6.

Figure 7

¹H NMR spectrum (CDCl₃, 300 MHz) of Z/E 6. In the red squares are evidenced significant selected regions of the spectrum (solid line) and the related magnification (dotted line). The red arrows indicated the signals related to the indicated groups.

Figure 8

¹³C NMR spectrum (CDCl₃, 75.5 MHz) of Z/E 6. In the red squares are evidenced significant selected regions of the spectrum and the related magnification. The purple bar groups the signals of the 20 methylene groups.

Figure 9

Potentiometric titration profiles of 1 (a) (green line) and 6 (b) (red line) and related first derivative curves (FD) (purple lines).

Figure 10

Time-killing curves performed with 1 (at concentrations equal to 4 × MIC) on S. aureus 18.

Figure 11

Growth inhibiting effects of QPS 1 on HepG2 cells. HepG2 cells were seeded on 96-multiwell plates and treated for 24 h with different concentrations (2–160 µg/mL) of the QPS 1 compound. Cell viability was determined by MTT assay. (a) The bar graph shows the cell viability (%) of the HepG2 cells after exposure for 24 h to no-DMSO (CTRL, control conditions), DMSO (1 µL ml ⁻¹), and increasing concentrations (2–160 µg/mL) of QPS 1. Data are expressed as the mean ± S.D. of the survival percentage obtained from n = 5–6 independent experiments run in triplicate. Significance is indicated as *** p < 0.0001 and n.s. (no statistical difference) vs. CTRL (one-way ANOVA followed by Tukey’s multi-comparisons test, F _(4,20) = 61.23). (b) Representative images of HepG2 cells and formazan crystal formation after 2h of MTT exposure in DMSO- or QPS 1-treated cells; scale bars 20 µm.

Figure 12

Growth-inhibiting effects of QAS 6 on HepG2 cells. HepG2 cells were seeded on 96-multiwell plates and treated for 24 h with different concentrations (2–160 µg/mL) of the QAS 6 compound. Cell viability was determined by the MTT assay. (a) The bar graph shows the cell viability (%) of the HepG2 cells after exposure for 24 h to no-DMSO (CTRL, control conditions), DMSO (2 µL mL⁻¹), and increasing concentrations (8–640 µg/mL) of QAS 6. Data are expressed as the mean ± S.D. of the survival percentage obtained from n = 5–7 independent experiments run in triplicate. Significance is indicated as *** p < 0.0001 and n.s. (no statistical difference) vs. CTRL (one-way ANOVA followed by Tukey’s multi-comparisons test, F _(4,20) =61.23). (b) Representative images of HepG2 cells and formazan crystal formation after 2 h of MTT exposure in DMSO- or QAS 6-treated cells; scale bars: 20 µm.