Dietary modulation of the resistance to intestinal infections.

Gastrointestinal infections are still a major health problem, not only in developing countries. Even in Europe and the United States about 10-15 % of the population contracts an intestinal infection each year, mostly of foodborne origin. The growing resistance of pathogens to antibiotics stresses the importance to prevent and treat intestinal infections by other means. Modulation of the diet to improve host resistance to foodborne infections might be an attractive, alternative approach.The diet determines the composition of intestinal contents, which in turn affects the gastrointestinal survival of pathogens, the protective endogenous microflora and the epithelial barrier function. These parameters ultimately determine the susceptibility of the host to intestinal infectious disease. Scientific interest in dietary modulation of the resistance to intestinal infections is just emerging. Notwithstanding the results of numerous in-vitro studies, strictly controlled infection studies showing the importance of the diet (supplemented with pre- or probiotics) to inhibit or ameliorate intestinal infections in-vivo are scarce. Even less is known about the potential protective effect of dietary calcium on the resistance to intestinal infections. At the same time, evidence accumulates showing that calcium is likely an antipromoter of colon carcinogenesis.In the intestine, calcium forms an insoluble complex with phosphate which strongly binds bile acids and fatty acids. In soluble form, these surfactants are highly irritating to the intestinal epithelium. Therefore, precipitation of bile acids and fatty acids by calcium phosphate decreases luminal cytotoxicity, resulting in diminished epithelial cell damage and reduced epithelial proliferation. This may also be relevant for host resistance to intestinal infections. A reduced epithelial cell damage may strengthen the mucosal barrier function. In addition, it can be speculated that the decreased cytotoxicity of intestinal contents by calcium phosphate may stimulate growth of the protective endogenous microflora and improve its antagonistic activity towards invading pathogens. Figure 1 summarizes the hypothetical mechanism by which dietary calcium phosphate may decrease the severity of an intestinal infection, for instance caused by salmonella.The strictly-controlled experimental studies described in this thesis mainly focused on the proposed protective effect of dietary calcium phosphate on the resistance to intestinal infections. The rat was chosen as animal model and the invasive pathogen Salmonella enteritidis as infective agent. Salmonellosis is one of the most common foodborne, bacterial infections in the world and its pathology in humans and rodents is quite similar. The first study of this thesis investigated the application of urinary nitrate excretion as a marker of intestinal bacterial translocation (chapter 2). To study dietary modulation of host resistance to translocation of pathogens a non-invasive, sensitive and quantifiable marker is needed. Classical organ cultures do not meet those criteria. Nitric oxide (NO) is produced by inducible nitric oxide synthase of phagocytes upon contact with bacteria or cell wall components of bacteria, like lipopolysaccharides. To prevent damage to host cells, NO is rapidly oxidized to nitrite and nitrate (summed as NO x ) and these are quantitatively excreted in urine. It was shown that intraperitoneally injected S. enteritidis lipopolysaccharides transiently increased the urinary NO x output within a certain dose-range. Concomitant administration of a competitive inhibitor of nitric oxide synthase (N G -nitro-L-arginine methyl ester) almost completely abolished the rise in NO x excretion. Importantly, increasing the oral dose of viable S. enteritidis resulted in a time- and dose-dependent exponential increase in urinary NO x excretion. Translocation was a prerequisite for provoking a NO x response, because neither orally administered, heat-killed S. enteritidis nor non-invasive, enterotoxigenic Escherichia coli (data not shown) induced an increase in NO x excretion above base-line level. Total urinary NO x excretion after infection of the rats with viable S. enteritidis and weight of the mesenteric lymph nodes were highly correlated.After validation of this new translocation marker, the effect of different milk products (low-calcium milk, milk, milk acidified with hydrochloric acid, and pasteurized yogurt) on the resistance of rats to S. enteritidis was studied (chapter 3). Compared with the low-calcium milk group, all high-calcium groups had an increased colonization resistance, as judged by the strongly reduced fecal salmonella excretion in time. The yogurt-fed rats had the best colonization resistance. Before infection, the bile acid concentration and cytotoxicity of fecal water of the low-calcium milk group were significantly higher than those of the high-calcium groups. The reduced resistance of the low-calcium milk group corresponded with strong infection-induced disturbances of normal intestinal physiology. For instance, the apparent iron absorption was reduced and considerable increases in cytotoxicity of fecal water, fecal mucin and alkaline phosphatase excretion were observed in this group. The least infection-induced changes in luminal parameters were noticed in the yogurt-fed rats. Surprisingly, no infection-induced increase in urinary NO x excretion was observed in this study (data not shown). As the milk-based diets differed in several respects, another strictly controlled infection study was performed with rats on purified diets differing only in calcium phosphate (20, 60 and 180 mmol/kg) content (chapter 4). Compared with the low-calcium group, the medium- and high-calcium group shedded 10-1000 times less salmonella in their feces and thus had a substantially improved colonization resistance. Calcium supplementation also reduced translocation of salmonella, considering the diminished urinary NO x excretion and decreased viable salmonella counts in the ileal Peyer's patches and spleen. As shown earlier, the bile acid concentration and cytotoxicity of fecal water were decreased by dietary calcium phosphate. This resulted in an increased fecal output of several bacterial mass markers, indicating a stimulation of the endogenous microflora. Besides an enhanced fecal dry weight excretion, dietary calcium phosphate also increased fecal nitrogen, phospholipid and organic phosphate output.The non-digestible disaccharide lactulose is well-fermented by the intestinal microflora and has been used successfully in the treatment of certain intestinal infections. The organic acids (e.g. lactic acid) formed during bacterial lactulose fermentation probably play an important role in this protection. Nevertheless, excess acid production may damage the intestinal epithelium and even impair the mucosal barrier function. Considering the above-mentioned resistance-enhancing effects of dietary calcium phosphate and its ability to increase the intestinal buffering capacity, the possible superiority of a combination of dietary lactulose and calcium phosphate to improve host resistance was studied (chapter 5). S. enteritidis appeared to be very sensitive to lactic acid in-vitro, whereas Lactobacillus acidophilus (as a representative of the protective endogenous microflora) was unaffected. The infection experiment showed that dietary lactulose decreased fecal shedding of salmonella, thus increased the colonization resistance. The protective effects of lactulose were limited to the cecum and colon because this disaccharide did not decrease translocation of salmonella, as measured by urinary NO x excretion. In agreement with the study described above, calcium phosphate significantly inhibited translocation of salmonella. It is known that mucosal invasion of salmonella mainly takes place in the ileum, a region of the intestinal tract with a relatively less dense bacterial population. Obviously, the fermentation of lactulose in the ileum is limited and not sufficient to prevent translocation of salmonella. Supplementation of a lactulose diet with calcium phosphate reversed the unfavorable increased cytotoxicity of fecal water. In addition, calcium phosphate stimulated lactulose fermentation, as judged by the reduced lactulose excretion in feces and increased fecal lactic acid, ammonia, and nitrogen excretion.Finally, it was investigated whether the calcium phosphate-induced protection against colonization and translocation of salmonella was mediated by a stimulation of the intestinal lactobacilli (chapter 6). In-vitro, L. acidophilus was rapidly killed by physiologically relevant concentrations of fatty acids and (un)conjugated bile acids. In contrast, even high concentrations of these surfactants did not affect the viability of S. enteritidis . Calcium phosphate-supplementation reduced the cytotoxicity and the concentration of bile acids and fatty acids in ileal contents and fecal water of rats. Moreover, calcium phosphate notably changed the composition of ileal bile acids into a less cell-damaging direction. Consequently, significantly increased numbers of lactobacilli were detected in ileal contents, on the ileal mucosa and in feces of non-infected, calcium phosphate-supplemented animals. At the same time, the calcium phosphate group had less viable salmonella in ileal contents, on the ileal mucosa and in feces. In accordance, the infection-induced urinary NO x excretion was diminished by calcium phosphate supplementation.