RESUMO
The gut microbiota plays an important role in host health, in particular by its barrier effect and competition with exogenous pathogenic bacteria. In the present study, the competition of Bifidobacterium pseudolongum PV8-2 (Bp PV8-2) and Bifidobacterium kashiwanohense PV20-2 (Bk PV20-2), isolated from anemic infant gut microbiota and selected for their high iron sequestration properties, was investigated against Salmonella Typhimurium (S. Typhi) and Escherichia coli O157:H45 (EHEC) by using co-culture tests and assays with intestinal cell lines. Single and co-cultures were carried out anaerobically in chemically semi-defined low iron (1.5 µM Fe) medium (CSDLIM) without and with added ferrous iron (30 µM Fe). Surface properties of the tested strains were measured by bacterial adhesion to solvent xylene, chloroform, ethyl acetate, and to extracellular matrix molecules, mucus II, collagen I, fibrinogen, fibronectin. HT29-MTX mucus-secreting intestinal cell cultures were used to study bifidobacteria competition, inhibition and displacement of the enteropathogens. During co-cultures in CSDLIM we observed strain-dependent inhibition of bifidobacterial strains on enteropathogens, independent of pH, organic acid production and supplemented iron. Bp PV8-2 significantly (P < 0.05) inhibited S. Typhi N15 and EHEC after 24 h compared to single culture growth. In contrast Bk PV20-2 showed less inhibition on S. Typhi N15 than Bp PV8-2, and no inhibition on EHEC. Affinity for intestinal cell surface glycoproteins was strain-specific, with high affinity of Bp PV8-2 for mucin and Bk PV20-2 for fibronectin. Bk PV20-2 showed high adhesion potential (15.6 ± 6.0%) to HT29-MTX cell layer compared to Bp PV8-2 (1.4 ± 0.4%). In competition, inhibition and displacement tests, Bp PV8-2 significantly (P < 0.05) reduced S. Typhi N15 and EHEC adhesion, while Bk PV20-2 was only active on S. Typhi N15 adhesion. To conclude, bifidobacterial strains selected for their high iron binding properties inhibited S. Typhi N15 and EHEC in co-culture experiments and efficiently competed with the enteropathogens on mucus-producing HT29-MTX cell lines. Further studies in complex gut ecosystems should explore host protection effects of Bp PV8-2 and Bk PV20-2 mediated by nutritional immunity mechanism associated with iron-binding.
RESUMO
The presence of iron(II) in beta-glucan in solution causes the formation of hydroxyl radical, which further oxidises the polysaccharide. This degradation can be enhanced by the presence of a reducing agent, such as ascorbic acid. In this study we investigated the effect the iron(II) concentration on the hydroxyl radical-mediated degradation of beta-glucan and identified the intermediate species involved in the formation of hydroxyl radicals. An increase in the iron(II) concentration did not have a significant effect on the degradation in the presence of a reducing agent (ascorbic acid), while in the mere presence of iron(II) it accelerates the degradation. The addition of catalase and superoxide dismutase (SOD) prevented the hydroxyl radical driven-degradation of beta-glucan induced by iron(II) or ascorbic acid/iron(II), demonstrating the involvement of both superoxide and hydrogen peroxide in the hydroxyl radical formation. SOD, which catalyses the dismutation of superoxide into hydrogen peroxide, should have stimulated the formation of radicals, since these radicals are generated from the reaction between hydrogen peroxide and iron(II). In the present study, we hypothesise the mechanism of the inhibition of beta-glucan degradation by superoxide dismutase.
