Abstract
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.
Highlights
Bifidobacteria are among the first commensal anaerobic bacteria that reach high levels in the infant gut within the first week of life, representing up to 50–80% of the gut bacteria (Jost et al, 2012; Turroni et al, 2012)
The gut microbiota is constantly challenged by different stress factors, including enteropathogens, such as Salmonella and Escherichia coli O157:H45 (EHEC; Wardlaw et al, 2010)
In a previous study we reported isolation of 56 bifidobacterial strains from stools of breast fed, iron-deficient and anemic Kenyan infants (VazquezGutierrez et al, 2015c)
Summary
Bifidobacteria are among the first commensal anaerobic bacteria that reach high levels in the infant gut within the first week of life, representing up to 50–80% of the gut bacteria (Jost et al, 2012; Turroni et al, 2012). The establishment of bifidobacteria in the gut has been associated with a broad range of beneficial effects on host health, such as modulation of intestinal microbiota composition, prevention of infection and immune-modulation (Broekaert and Walker, 2006; Yatsunenko et al, 2012). Inhibition of pathogens in the gut by bifidobacteria might be due to production of inhibitory substances, inhibition of epithelial and mucosal invasion of pathogens, competition for limited nutrients and/or the stimulation of mucosal immunity (Marco et al, 2006; Turroni et al, 2014). Bifidobacteria can compete with pathogens for adhesion to intestinal epithelial sites and nutrients, enhancing resistance to colonization of pathogenic bacteria (Collado et al, 2007; Aires et al, 2010). To inhibit pathogen infection in the gut, commensal intestinal microorganisms such as bifidobacteria, should be able to compete for corresponding niches. The inhibitory activity and mechanisms of bifidobacteria against enteropathogens have been investigated by microbe-microbe and cell-microbe interaction models (Collado et al, 2007)
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