Pistacia vera L. oleoresin and levofloxacin is a synergistic combination against resistant Helicobacter pylori strains

Abstract

The increasing multidrug resistance in Helicobacter pylori, also correlated to its biofilm-forming ability, underlines the need to search novel strategies to improve the eradication rate. Natural compounds are proposed as antibiotic-resistant-breakers capable to restore the efficacy of conventional drugs. Aim of this work was to evaluate the capability of Pistacia vera L. oleoresin (ORS) to synergize with levofloxacin (LVX) against resistant H. pylori strains. The antimicrobial activity of P. vera L. ORS and LVX and their combinations was determined by MIC/MBC (in neutral and acidic environments) and checkerboard tests. The anti-biofilm effect was determined by biomass quantification. In vivo Galleria mellonella model was used to confirm in vitro data. Pistacia vera L. ORS and LVX MICs ranged respectively from 780 to 3120 mg/l and from 0.12 to 2.00 mg/l, at pH 7.0 and 5.5. MBCs were similar to MICs. Pistacia vera L. ORS was able to synergize with LVX, restoring its effectiveness in LVX resistant strains. Pistacia vera L. ORS, LVX and their synergistic combinations displayed significant biofilm reduction. Pistacia vera L. ORS and LVX, showed protective effect against H. pylori infection on G. mellonella (62% and 63% of survival, respectively). Pistacia vera L. ORS can be considered a promising potentiator to restore the effectiveness of LVX tackling the H. pylori antibiotic resistance phenomenon.

Figure 1

Representative images of checkerboard assays and isobolograms for evaluation of synergism between P. vera L. ORS and LVX combinations against H. pylori 2A/12 and H. pylori 11F/11. The calculation of the best P. vera L. ORS and LVX combination and the Fractional Inhibitory Concentration Index (FIC I) values were interpreted as follow: synergism (FIC I ≤ 0.5), antagonism (FIC I ≥ 4.0), and additive effect (FIC I > 0.5–4.0). For H. pylori 2A/12, the MIC values are 1560 mg/l and 0.50 mg/l for P. vera L. ORS and LVX, respectively. For H. pylori 11F/11, the MIC values are 1560 mg/l and 1.00 mg/l for P. vera L. ORS and LVX, respectively. The grey zone represents the bacterial growth and the white zone represents the inhibition of bacterial growth in presence of P. vera L. ORS and LVX. The synergistic combinations of P. vera L. ORS and LVX are the white zone in which FIC I is ≤0.5. On the right, the isobolograms illustrate the result of the checkerboard assay and the FIC I values, showing the synergistic curve. The x axis of the isobologram represents the dose of LVX and the y axis represents the dose of P. vera L. ORS. The imaginary straight line connecting the intercept points represents no interaction. Between this line and the synergistic curve, there is the area of synergistic (FIC I ≤ 0.5) and additive (FIC I > 0.5–4.0) interactions. Values above of the straight line represent antagonistic interactions (FIC I ≥ 4.0).

Figure 2

Representative images of Live/Dead staining of H. pylori 11F/11 biofilms. (A,B), control images (viable cells embedded in abundant biofilm were highlighted in A and viable rod shaped cells in the biofilm were highlighted in B). (C), biofilm treated with 1/4 MIC of P. vera L. ORS (prevalent viable coccoid cells in small aggregates). (D), biofilm treated with 1/8 MIC of LVX (prevalent dead rod cells without considerable aggregation). (E,F), biofilm treated with sub-synergistic concentrations of P. vera L. ORS and LVX showing their combined anti-adhesive and killing effect. (E), biofilm treated with 1/8 synergistic concentration of P. vera L. ORS (190 mg/l) and LVX (0.12 mg/l). (F), biofilm treated with 1/16 synergistic concentration of P. vera L. ORS (90 mg/l) and LVX (0.25 mg/l). Sessile population in biofilms stained in red (Propidium iodide) shows a damaged membrane (dead cells), whereas green stained bacteria (SYTO 9) represent viable cells. The images observed at fluorescent Leica 4000 DM microscopy (Leica Microsystems, Milan, Italy) were recorded at an emission wave length of 500 nm for SYTO 9 and of 635 nm for Propidium iodide and more fields of view randomly were examined. Original magnification, 1000X.

Figure 3

In vivo infection assay in Galleria mellonella model. (A) Survival of G. mellonella after H. pylori 11F/11 infection with PBS, MIC of LVX, 1000 mg/kg of P. vera L. ORS and the best synergistic combination of P. vera L. ORS and LVX (90 mg/l ORS + 0.12 mg/l LVX, FIC I = 0.18). Kaplan-Meier survival curves of G. mellonella larvae after infection with 1.8 × 10⁶ CFUs. Differences in survival were calculated using the Long-rank test for multiple comparison. (B) Recovery of H. pylori 11F/11 CFU/larva in G. mellonella larvae after injection of 1.8 × 10⁶ CFUs after different time points and at different conditions (PBS, LVX at MIC value, P. vera L. ORS at 1000 mg/kg, the best synergistic combination of P. vera L. ORS and LVX).*Statistically significant in respect to sham injection, at each time control (p ≤ 0.05).