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. 2012 Sep 4;9(9):2677-85.
doi: 10.1021/mp300243w. Epub 2012 Aug 6.

Antibacterial Activities of Liposomal Linolenic Acids Against Antibiotic-Resistant Helicobacter Pylori

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Free PMC article

Antibacterial Activities of Liposomal Linolenic Acids Against Antibiotic-Resistant Helicobacter Pylori

Marygorret Obonyo et al. Mol Pharm. .
Free PMC article

Abstract

Helicobacter pylori (H. pylori) infection with its vast prevalence is responsible for various gastric diseases including gastritis, peptic ulcers, and gastric malignancy. While effective, current treatment regimens are challenged by a fast-declining eradication rate due to the increasing emergence of H. pylori strains resistant to existing antibiotics. Therefore, there is an urgent need to develop novel antibacterial strategies against H. pylori. In this study, we developed a liposomal nanoformulation of linolenic acid (LipoLLA) and evaluated its bactericidal activity against resistant strains of H. pylori. Using a laboratory strain of H. pylori, we found that LipoLLA was effective in killing both spiral and coccoid forms of the bacteria via disrupting bacterial membranes. Using a metronidazole-resistant strain of H. pylori and seven clinically isolated strains, we further demonstrated that LipoLLA eradicated all strains of the bacteria regardless of their antibiotic resistance status. Furthermore, under our experimental conditions, the bacteria did not develop drug resistance when cultured with LipoLLA at various sub-bactericidal concentrations, whereas they rapidly acquired resistance to both metronidazole and free linolenic acid (LLA). Our findings suggest that LipoLLA is a promising antibacterial nanotherapeutic to treat antibiotic-resistant H. pylori infection.

Figures

Figure 1
Figure 1
A schematic drawing shows the molecular structure of LLA and the structure of LipoLLA composed of phospholipid, cholesterol and LLA.
Figure 2
Figure 2
In vitro bactericidal activity of (A) LLA and (B) LipoLLA at different drug concentrations against H. pylori SS1. All concentrations refer to LLA concentration, regardless of the formulation. LLA or LipoLLA was incubated with 5×106 CFU/mL H. pylori SS1 bacteria at 37°C under microaerobic conditions for 30 min followed by Columbia agar plate subculture.
Figure 3
Figure 3
FRET measurements of the fusion between LipoLLA and H. pylori SS1. A fluorescent donor (C6NBD, 0.1 mol%) and a fluorescent acceptor (DMPE-RhB, 0.5 mol%) were concurrently incorporated into the lipid bilayer membranes of LipoLLA so that the acceptor completely quenched the fluorescence emission from the donor. The FRET-pair labeled LipoLLA was incubated with H. pylori at a concentration of a–e: 8.0×107, 1.6×108, 2.4×108, 3.2×108, and 4×108 CFU/mL for 10 min. After removing the excess LipoLLA, all samples were excited at 470 nm. A rise in emission intensity of C6NBD (donor) at 520 nm was observed with the increase of bacterial concentrations, indicating the occurrence of fusion between LipoLLA and H. pylori that caused the spatial separation of C6NBD and DMPE-RhB.
Figure 4
Figure 4
In vitro bactericidal activity of LipoLLA in comparison with LLA and Amoxicillin against (A) spiral form and (B) coccoid form of H. pylori SS1. Bacterial viability was tested by using Alarmar blue dye assay after an incubation period of 0.5, 4, or 24 hr. All concentrations of LLA and LipoLLA refer to LLA concentration, regardless of the formulation. Bacterial viability was normalized to the control sample treated with PBS buffer.
Figure 5
Figure 5
Morphology of H. pylori SS1 bacteria in their spiral form (A–C) and coccoid form (D–F) exposed to different treatments. In (A) and (D), bacteria were treated with PBS; in (B) and (E), bacteria were treated with LLA pre-dissolved in DMSO; in (C) and (F), bacteria were treated with LipoLLA. In all experiments, initial concentration of the bacteria was 5×106 CFU/mL and drug concentration was 200 µg/mL (referring to LLA). All samples were treated for 30 min before glutaralderhyde fixation. The scale bar in the image represents 1 µm.
Figure 6
Figure 6
In vitro bactericidal activity of (A) LLA and (B) LipoLLA against various H. pylori clinical isolates and a metronidazole-resistant strain of H. pylori SS1 (Mtzr SS1 mutant). In all experiments, 5×106 CFU/mL bacteria were incubated with 0, 100, or 200 µg/mL LLA, either as free LLA pre-dissolved in DMSO or in liposome formulation (LipoLLA). Samples were incubated at 37°C under microaerobic conditions for 30 min before serial dilution followed by bacterial colony enumeration on Columbia agar plates.

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