1 Division of Microbiology and Risk Assessment, National Food Institute, Technical University of Denmark2 National Food Institute, Technical University of Denmark3 Division of Toxicology and Risk Assessment, National Food Institute, Technical University of Denmark4 Copenhagen Center for Health Technology, Center, Technical University of Denmark
The human gastrointestinal tract is colonized by a dense and complex community of bacteria. The intestinal microbiota has a large impact on the health of the host and the intestinal bacteria are roughly classified as either potential deleterious or potential beneficial bacteria. Several factors can affect the composition of the microbiota - among them prebiotics. Prebiotics are food ingredients that are non-digestible for the human body and therefore reach the large intestine in an intact form. In the large intestine the prebiotics selectively stimulate the growth of the beneficial rather than the harmful bacteria of the microbiota. Gastro-intestinal infections currently cause several hundred thousand reported cases of disease in the EU each year. Infections with the forborne pathogen Listeria monocytogenes are relatively rare, but it is one of the most severe infections in the industrialised countries with a mortality of about 30%. The gut has a very important function in defending the host against infections with ingested pathogenic bacteria and there is increasing evidence that prebiotics can help strengthen this defense. This is done through stimulation of beneficial intestinal bacteria that release bacteriocins toxic for the pathogens, lower the pH to a level that is unfavourable for pathogenic bacteria and compete with the pathogen for nutrients and mucosal adhesion sites in the intestine. However, besides the microbiota dependent mechanisms increasing evidence suggest that prebiotics exert their protective function against pathogens through microbiota independent mechanisms. This is thought to be done by blocking the pathogenic adhesion to intestinal cells, affecting the expression of virulence genes from the pathogen and by stimulating the immune system. In vivo evidence of the prebiotics effect against pathogenic enteric infections is scarce and I therefore investigated the effect of five non-digestible carbohydrates (putative prebiotics) on the resistance of guinea pigs to infection with three different strains of L. monocytogenes. Animals were fed a diet supplemented with either 10% xylooligosaccharides (XOS), galactooligosaccharides (GOS), inulin, apple pectin or polydextrose for three weeks before oral challenge with L. monocytogenes. XOS and GOS significantly improved resistance of guinea pigs to L. monocytogenes, while inulin and apple pectin decreased the resistance. No significant effect on resistance to L. monocytogenes was seen after feeding with polydextrose. To further explore the mechanisms behind these in vivo observations, microbiota independent effects of four of the carbohydrates (XOS, GOS, inulin and polydextrose) on the adhesive and infective potential of L. monocytogenes was investigated. Mixing L. monocytogenes with XOS just prior to infection decreased the adherence of two of the three strains of L. monocytogenes to the intestinal cell line Caco-2. Additionally, 2 hours incubation with XOS and subsequently washing of the bacteria decreased the adherence of all three strains of L. monocytogenes to Caco-2 cells. No effect on adhesion was seen for either GOS, inulin or polydextrose. Adherence to the intestinal epithelium is considered a very important step in the infection cycle for most of the pathogenic bacteria. Without adherence the pathogenic bacteria are rapidly eliminated from the intestine. The ability of the four carbohydrates to affect the expression of L. monocytogenes genes known to be involved in adherence to intestinal cells (inlA, lap, ami, iap, aut, fdpA, actA) was therefore investigated. It was found that expression of the adhesion genes was affected in a strain dependent manner by the presence of prebiotics in the growth media.In conclusion, these results show that different non-digestible carbohydrates can have entirely different effects on the in vivo infectivity of L. monocytogenes and that microbiota independent mechanisms might be involved. All the tested carbohydrates affected expression of adherence genes but only XOS affected the in vitro adhesion of L. monocytogenes to intestinal cell. This may suggest that different mechanisms are responsible for the observed in vivo effect of the different non-digestible carbohydrates. Mostly microbiota independent mechanisms were investigated in this project, but it is very likely that microbiota dependent mechanisms also are involved.