Lippia graveolens Essential Oil to Enhance the Effect of Imipenem against Axenic and Co-Cultures of Pseudomonas aeruginosa and Acinetobacter baumannii

Abstract

This research focuses on assessing the synergistic effects of Mexican oregano (Lippia graveolens) essential oil or carvacrol when combined with the antibiotic imipenem, aiming to reduce the pathogenic viability and virulence of Acinetobacter baumannii and Pseudomonas aeruginosa. The study highlighted the synergistic effect of combining L. graveolens essential oil or carvacrol with imipenem, significantly reducing the required doses for inhibiting bacterial growth. The combination treatments drastically lowered the necessary imipenem doses, highlighting a potent enhancement in efficacy against A. baumannii and P. aeruginosa. For example, the minimum inhibitory concentrations (MIC) for the essential oil/imipenem combinations were notably low, at 0.03/0.000023 mg/mL for A. baumannii and 0.0073/0.000023 mg/mL for P. aeruginosa. Similarly, the combinations significantly inhibited biofilm formation at lower concentrations than when the components were used individually, demonstrating the strategic advantage of this approach in combating antibiotic resistance. For OXA-51, imipenem showed a relatively stable interaction during 30 ns of dynamic simulation of their interaction, indicating changes (<2 nm) in ligand positioning during this period. Carvacrol exhibited similar fluctuations to imipenem, suggesting its potential inhibition efficacy, while thymol showed significant variability, particularly at >10 ns, suggesting potential instability. With IMP-1, imipenem also displayed very stable interactions during 38 ns and demonstrated notable movement and positioning changes within the active site, indicating a more dynamic interaction. In contrast, carvacrol and thymol maintained their position within the active site only ~20 and ~15 ns, respectively. These results highlight the effectiveness of combining L. graveolens essential oil and carvacrol with imipenem in tackling the difficult-to-treat pathogens A. baumannii and P. aeruginosa.

Keywords: A. baumannii; Lippia graveolens; P. aeruginosa; carvacrol; co-cultures.

Figure 1

Fluorescence microscopy (200×) of biofilms of A. baumannii, P. aeruginosa and their co-culture on glass coverslips, incubated at 37 °C for 24 h in LB broth exposed to ½ MIC of the treatments essential oil of L. graveolens (EO), carvacrol and combinations with imipenem, stained with Syto 9 at 0.001%. (a) Control A. baumannii, (b) control P. aeruginosa, (c) co-culture control, (d) biofilm of A. baumannii exposed to 0.312 mg/mL EO, (e) biofilm of P. aeruginosa exposed to 0.078 mg/mL of EO, (f) co-culture biofilm exposed to 0.625 mg/mL of EO, (g) biofilm of A. baumannii exposed to 0.075 mg/mL of carvacrol, (h) biofilm of P. aeruginosa exposed to 0.0375 mg/mL carvacrol, (i) co-culture biofilm exposed to 0.15 mg/mL carvacrol, (j) A. baumannii biofilm exposed to EO–imipenem combination of 0.015/1.17 × 10⁻⁵ mg/mL, (k) P. aeruginosa biofilm exposed to EO–imipenem combination of 3.65 × 10⁻³/1.17 × 10⁻⁵ mg/mL, (l) co-culture biofilm exposed to EO–imipenem combination of 0.03/2.34 × 10⁻⁵ mg/mL, (m) biofilm of A. baumannii exposed to the carvacrol–imipenem combination of 3.5 × 10⁻³/1.17 × 10⁻⁵ mg/mL, (n) biofilm of P. aeruginosa biofilm exposed to the carvacrol–imipenem combination of 1.75 × 10⁻³/2.34 × 10⁻⁵ mg/mL, (o) co-culture biofilm exposed to the carvacrol–imipenem combination of 0.007/2.34 × 10⁻⁵ mg/mL.

Figure 2

Deviation of the root mean square deviation of the atomic positions (RMSD) of imipenem, carvacrol and thymol and its binding free energy (Kcal/mol) with β-lactamase OXA-51 ((a) and (b), respectively) and IMP-1 (c,d). The behavior of imipenem is shown in black (), carvacrol in red () and thymol in blue ().