Ir J Med Sci. 2013 Sep;182(3):439-52.

Lactic acid bacterial extracts as anti-Helicobacter pylori: a molecular approach.

El-Adawi H, El-Sheekh M, Khalil M, El-Deeb N, Hussein M.

Department of Medical Biotechnology, Genetic Engineering and Biotech Institute, Mubarak City for Scientific Research, Universities and Research District Sector, New Borg El-Arab, P.O.BOX 29134, Alexandria, Egypt. halaeladawi@hotmail.com

 

Abstract

Background: Helicobacter pylori (H. pylori) infection, the main cause of chronic gastritis, increases gastric cancer risk. Antibiotics-based H. pylori eradication treatment is 90 % effective. However, it is expensive and causes side effects and antibiotic resistance. Lactic acid bacteria (LAB) could present a low-cost, large-scale alternative solution to prevent or decrease H. pylori colonization.

Aim: This work aimed to study the inhibitory effects of LAB strains on the growth and pathogenic activity of H. pylori stains. To this end, we have selected the most virulent H. pylori strains (out of 20 mucosal antral biopsies) regarding cellular vacuolization and induction of apoptosis/necrosis.

Method The selection of H. pylori pathogenic strains (clinically pre-isolated) were based on their impact of VacA activities on Hep-2 cell line, induction of apoptosis and necrosis in Caco-2 cell line. The Inhibitory effect of LAB strains on the invasion was carried out using the Caco-2 and Hela cell lines, where, they were co-cultured with the pathogenic H. pylori in the presence or absence of LAB extracts. The effect of LAB extracts on TNF-a secretion which induced by H. pylori-LPS was carried out by RT-qPCR.

Results: L. bulgaricus DSMZ 20080, L. acidophilus and L. plantarum (studied previously and reported as high antioxidant candidate strains) showed the highest antipylori activities with inhibition ranged from 51.46 to 88.19 %, they preventing the adhesion, invasion and DNA fragmentation of cell lines. In addition, they could reduce the TNF-a expression by 62.13 %.

Conclusion : LAB extracts could inhibit the bacterial adhesion and invasion, gastric inflammation and DNA fragmentation induced by Helicobacter pylori.

PMID: 23404362

 

Supplements:

Helicobacter pylori (H. pylori) are spiral-shaped bacteria that colonize the stomach of half the populations worldwide. Once a person is infected, the organism can live in the stomach indefinitely. Although H. pylori infection is a common infection, it is not benign. Close contact of H. pylori with the epithelium results in the secretion of free radicals and cytokines such as interleukin-8 IL-8, IL-1and a-TNF. Excessive cytokines secretion is considered to play an important role in the alteration of epithelial proliferation, increased apoptosis oxidative DNA damages, occurrence of chronic gastritis, gastric lesions and duodenal and gastric ulcers that are the strongest known risk factors for adenocarcinoma of the distal stomach. Prevalence of H. pylori antibiotic resistance is increasing worldwide, and it is the main factor affecting efficacy of current therapeutic regimens. We hypothesized that Lactic acid bacteria (LAB) could present a low cost, large-scale alternative solution to prevent or decrease H. pylori colonization.

Our laboratory studied previously and reported L. bulgaricus DSMZ 20080, L. acidophilus and L. plantarum as high antioxidant LAB candidate strains [1]. Our work aimed to study the inhibitory effects of LAB strains on the growth and pathogenic activity of H. pylori strains. To this end, we have selected the most virulent H. pylori strains (out of 20 mucosal antral biopsies) regarding cellular vacuolization (VacA) and induction of apoptosis/necrosis. Vacuolating activity results indicated that the ability of the isolates to induce vacuoles in epithelial cell lines ranged from weak and moderate to high ( Fig. 1).

fig1
Fig.1. Vaculating activity of the H. pylori clinical isolates on Hep-2 cells

 

In addition to the vacuolating activity, all isolates had the ability to induce all stages (early to late) of apoptosis and necrosis (Fig.2) fig2

Fig.2. Assessment of apoptosis and necrosis induced by Helicobacter pylori. Apoptotic cells had condensed and/or fragmented nuclei, these cells were impermeable to ethidium bromide during early stages and their nuclei stained green but during late stages, this ability was lasted and their nuclei stained red. Necrosis cells: cells exhibited a red nucleus stain but with no nuclear condensation they exhibited either normal nuclear structure or had no nuclear staining.

The antibacterial activity of LAB extracts indicated that, in general, the extracellular extracts of the LAB were more effective than the intracellular extract. The antibacterial activity of these treatments ranged from 81.44 to 88.19 %.

The pretreatment of H. pylori with the most potent antibacterial extracts prevents the remaining viable H. pylori from adhering to cell lines. In addition, after treatments, they conversed morphologically into a coccoid form either was viable but non-culturable or was killed, in comparison to the control group (in which the non-treated helical H. pylori were able to adhere to the cell lines). Visual inspection of Hep-2 cells after the infection/replication phases demonstrated internalized bacteria, sometimes enclosed in apparent vacuoles (Fig. 3).

fig3

Fig.3. Inhibition of H. pylori–Hep-2 adhesion by lactic acidbacteria. a, b Hep-2 mammalian cells infected with H. pylori. c Hep-2 mammalian cells infected with H. pylori treated with LAB. d Hep-2 mammalian cells (at high  magnification) infected with H. pylori treated with LAB. e Hep-2 mammalian cells (control). The small dark arrows in a, b pointed to H. pylori adhered to cell in the spiral shape, while in c, d pointed to the H. pylori under lactic acid bacterial treatmentconverted to coccid forms with reduction in their ability to adhered to cells.

 

Inhibition of H. pylori invasion to HepG2 cells by LAB was tested where, the HepG2 cells infected with H. pylori, green fluorescent bacteria, could be detected within HepG2 cells (indicating invasion) after staining with acridine orange (Fig. 4). The pretreatment of H. pylori with the most potent antibacterial extracts decreases the number of the invading bacteria from 20 cells to single H. pylori cell.

fig4

Fig. 4 Inhibition of H. pylori invasion to HepG2 cells by LAB; a the control HepG2 cells, b Hepg2 cells treated with LAB and infected with H. pylori, c H. pylori

 

We examined the possibility that H. pylori directly compromise the genomic integrity of its host cells. We provide evidence that the infection introduces DNA double-strand breaks (DSBs). The induction of DSBs depends on the direct contact of live bacteria with mammalian cells. The infection-associated DNA damage is evident upon separation of nuclear DNA (Fig. 5). fig5Fig. 5 Inhibitory effect of LAB on DNA fragmentation induced by H. pylori L ladder DNA. C DNA of the non-infected cell (control). I DNA of cells infected cells with H. pylori. T DNA of cells infected with H. pylori previously treated with LAB.

As shown in figure, the DNA of mammalian cell infected with H. pylori showed apoptotic DNA cleavage (characteristic oligonucleosomal fragments-lane I) whereas, in case of with the most potent antibacterial LAB treatment, DNA mammalian cell appears as non-segmented bands when compared with the infected one.

A large body of evidence has shown that the presence of H. pylori in contact with epithelium results in the secretion of various inflammatory response such as tumor necrosis factor (TNF-α). This inflammatory response due to the cell wall lipopolycaccharide (LPS) of H. pylori. In order to isolate H. pylori –LPS, a modified method by Uchida and Mizushima was carried out [2] and peripheral blood mononuclear cells ( PBMC) was cultured with the LPS stimulant  in the presence and absence of LAB extract. At the end of incubation period , the RNA extraction of PBMC was carried out and real- time RT-PCR analyses was done to reveal the effect of LAB extract on  TNF-α expression. Our results showed that the LAB extract could reduce the TNF-α expression by 62.13%.

Conclusion:

LAB extracts could inhibit the H. pylori adhesion, invasion and DNA fragmentation. In addition, it could modify the immunologic response of the host by modulating the secretion of anti-inflammatory cytokines, which would result in the reduction of gastric inflammation.

Acknowledgements: This work was supported by GEBRI-institute- City for Scientific Research, Alexandria –Egypt, and published in Irish journal of Medical Science 2013.

References:

1-      El-Adawi H, Khalil AM, El- Sheekh MM, El-Deeb NM, Hussein MZ(2012). Cytotoxicity assay and antioxidant activities of the Lactic acid bacterial strains. Afr J Microbil Res 6(8): 1700-1712.

2-      Uchida K, Mizushima S (1987). A simple method for isolation of lipopolysaccharide from Pseudomonas aeruginosa and some other bacterial strains. Agric Biol Chem 51:3107-3114

 

Hala El-Adawi, PhDfig6
Professor of Medical biotechnology
Dept. of Medical Biotechnology,
Genetic Engineering and Biotechnology Institute,
New Borg El-Arab, P.O. Box: 21934 Alexandria, Egypt
Tel:   +203-4593422
Fax: +203-4593423
http://www.mucsat.org/

 

 

 

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