High Concentrations of Sodium Chloride Improve Microbicidal Activity of Ibuprofen against Common Cystic Fibrosis Pathogens

Abstract

Ibuprofen (IBU-H), a widely used anti-inflammatory, also shows a marked antimicrobial effect against several bacterial species, including those involved in cystic fibrosis such as Pseudomona aeruginosa, methicillin resistant Staphylococcus aureus and Burkholderia cepacia complex. Additionally, our results show significant synergy between water soluble Na-ibuprofen (IBU-Na) and ionic strength. Salt concentrations above 0.5 M modify the zeta potential promoting the action of Na-IBU; thus, with 1 M sodium chloride, IBU-Na is ten times more efficient than in the absence of ionic strength, and the minimum effective contact time is reduced from hours to minutes. In short time periods, where neither IBU-Na nor controls with 1 M NaCl show activity, the combination of both leads to a reduction in the bacterial load. We also analyzed whether the changes caused by salt on the bacterial membrane also promoted the activity of other microbicide compounds used in cystic fibrosis like gentamicin, tobramycin and phosphomycin. The results show that the presence of ionic strength only enhanced the bactericidal activity of the amphipathic molecule of IBU-Na. In this respect, the effect of saline concentration was also reflected in the surface properties of IBU-Na, where, in addition to the clear differences observed between 145 mM and 1 M, singular behaviors were also found, different in each condition. The combination of anti-inflammatory activity and this improved bactericidal effect of Na-IBU in hypertonic solution provides a new alternative for the treatment of respiratory infections of fibrotic patients based on known and widely used compounds.

Keywords: P. aeruginosa; bactericide activity; cystic fibrosis; ibuprofen; synergy.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1

Minimum inhibitory concentration of IBU. (a) Bactericidal effect of increasing concentrations of IBU-Na and IBU-H (1, 5, 10, 25, 50, 100 mM) on the viability of P. aeruginosa at 1 × 106 cfu/mL after incubation for 24 h at 35 ± 2 °C. (b) Susceptibility of S. aureus, B. cepacia and P. aeruginosa at initial concentration of 1 × 106 cfu/mL to increasing concentrations of IBU-Na (1, 5, 10, 25, 50 mM) incubated at 35 ± 2 °C for 24 h.

Figure 2

Effect of incubation time on bactericidal activity of IBU. Bactericidal effect of IBU-Na between 1 to 100 mM concentration at different incubation times (1, 4 and 12 h) on P. aeruginosa inoculum (1 × 106 cfu/mL).

Figure 3

IBU-Na activity as a function of different initial inoculum titres. Effects of IBU-Na (1, 5, 10, 25 and 50 mM) were tested at three different initial inoculum densities referred to as Population A ♦-♦ (1 × 106 cfu/mL), B ■-■ (1 × 105 cfu/mL) and C ●-● (1 × 104 cfu/mL), obtained from decimal serial dilutions of P. aeruginosa 1 × 107 cfu/mL. The incubation was performed for 4 h at 37 °C.

Figure 4

Effect of ionic strength on the bactericidal effect of IBU. (a) Viability of P. aeruginosa incubated with IBU-Na (1, 5, 10, 25 and 50 mM) in the absence (▲) and presence (⚫)of 1 M NaCl 4 h at 35 ± 2 °C. (b) Effect of salt concentration on the bactericidal effect of IBU-Na. Viability of a suspension of P. aeruginosa 1 × 106 cfu/mL incubated for 60 min at 35 ± 2 °C with 10 mM IBU (○-○) as a function of salt concentration present in the medium. A control of NaCl effect without IBU (■-■) was assayed in similar conditions.

Figure 5

Effect of ionic strength on the microbicidal activity of different antibiotics. Effects of tobramycin, phosphomicine and gentamicin on viability of P. aeruginosa without (a) 1 M NaCl and (b) with 1 M NaCl.

Figure 6

IBU-Na adsorption at air-buffer (100 mM TRIS-HCl buffer pH 7.6) interface. Maximum surface pressure versus IBU-Na concentration (mM) with 145 mM (empty squares) and 1 M NaCl (full squares).

Figure 7

Effect of salt concentration on the absorption of IBU-Na. Surface pressure (at the air-buffer interface) versus time curves of increasing concentrations of IBU-Na with 145 mM (A) and 1 M NaCl (B).

Figure 8

Penetration of IBU-Na into bacterial phospholipid monolayers. (∆π)max of IBU-Na versus initial surface pressure of phospholipid monolayers with 145 mM (empty squares) and 1 M (full squares) NaCl.

Figure 9

Effect of salt concentration on the penetration of IBU-Na. Surface pressure versus time of IBU-Na injected into subphases of bacterial phospholipids with 145 mM and 1 M NaCl.

Figure 10

Penetration of IBU-Na into DOPE. (∆π)max of IBU-Na versus initial surface pressure of DOPE monolayers with 145 mM (empty squares) and 1 M (full squares) NaCl.