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.</p

Dietary calcium decreases but short-chain fructo-oligosaccharides increase colonic permeability in rats

An increased intestinal permeability is associated with several diseases. Nutrition can influence gut permeability. Previously, we showed that dietary Ca decreases whereas dietary short-chain fructo-oligosaccharides (scFOS) increase intestinal permeability in rats. However, it is unknown how and where in the gastrointestinal tract Ca and scFOS exert their effects. Rats were fed a Western low-Ca control diet, or a similar diet supplemented with either Ca or scFOS. Lactulose plus mannitol and Cr-EDTA were added to the diets to quantify small and total gastrointestinal permeability, respectively. Additionally, colonic tissue was mounted in Ussing chambers and exposed to faecal water of these rats. Dietary Ca immediately decreased urinary Cr-EDTA excretion by 24 % in Ca-fed rats compared with control rats. Dietary scFOS increased total Cr-EDTA permeability gradually with time, likely reflecting relatively slow gut microbiota adaptations, which finally resulted in a 30 % increase. The lactulose: mannitol ratio was 15 % higher for Ca-fed rats and 16 % lower for scFOS-fed rats compared with control rats. However, no dietary effect was present on individual urinary lactulose and mannitol excretion. The faecal waters did not influence colonic permeability in Ussing chambers. In conclusion, despite effects on the lactulose: mannitol ratio, individual lactulose values did not alter, indicating that diet did not influence small-intestinal permeability. Therefore, both nutrients affect permeability only in the colon: Ca decreases, while scFOS increase colonic permeability. As faecal water did not influence permeability in Ussing chambers, probably modulation of mucins and/or microbiota is important for the in vivo effects of dietary Ca and scFOS

Ileal Mucosal and Fecal Pancreatitis Associated Protein Levels Reflect Severity of Salmonella Inflection in Rats

Background Microbial infections induce ileal pancreatitis-associated protein/regenerating gene III (PAP/RegIII) mRNA expression. Despite increasing interest, little is known about the PAP/RegIII protein. Therefore, ileal mucosal PAP/RegIII protein expression, localization, and fecal excretion were studied in rats upon Salmonella infection. Results Salmonella infection increased ileal mucosal PAP/RegIII protein levels in enterocytes located at the crypt-villus junction. Increased colonization and translocation of Salmonella was associated with higher ileal mucosal PAP/RegIII levels and secretion of this protein in feces. Conclusions PAP/RegIII protein is increased in enterocytes of the ileal mucosa during Salmonella infection and is associated with infection severity. PAP/RegIII is excreted in feces and might be used as a new and non-invasive infection marke

Dietary fructo-oligosaccharides and latulose inhibit intestinal colonisation but stimulate translocation of salmonella in rats

Background and aims: It is frequently assumed that dietary non-digestible carbohydrates improve host resistance to intestinal infections by stimulating the protective gut microflora. However, compelling scientific evidence from in vivo infection studies is lacking. Therefore, we studied the effect of several non-digestible carbohydrates on the resistance of rats to Salmonella enteritidis infection. Methods: Rats (n = 8 per group) were fed "humanised'' purified diets containing 4% lactulose, fructo-oligosaccharides (FOS), resistant starch, wheat fibre, or cellulose. After an adaptation period of 2 weeks the animals were orally infected with S enteritidis. Supplement induced changes in faecal biochemical and microbiological parameters were studied before infection. Colonisation of salmonella was determined by studying the faecal excretion of this pathogen and translocation by analysis of urinary nitric oxide metabolites over time and classical organ cultures. Intestinal mucosal myeloperoxidase activity was determined to quantify intestinal inflammation after infection. Results: Despite stimulation of intestinal lactobacilli and bifidobacteria and inhibition of salmonella colonisation, FOS and lactulose significantly enhanced translocation of this pathogen. These supplements also increased cytotoxicity of faecal water and faecal mucin excretion, which may reflect mucosal irritation. In addition, caecal and colonic, but not ileal, mucosal myeloperoxidase activity was increased in infected rats fed FOS and lactulose. In contrast, cellulose, wheat fibre, and resistant starch did not affect the resistance to salmonella. Conclusions: In contrast to most expectations, FOS and lactulose impair the resistance of rats to intestinal salmonella infection. Obviously, stimulation of the endogenous lactobacilli and bifidobacteria is no guarantee of improved host defence against intestinal infections

The protective effect of supplemental calcium on colonic permeability depends on a calcium phosphate-induced increase in luminal buffering capacity

An increased intestinal permeability is associated with several diseases. Previously, we have shown that dietary Ca decreases colonic permeability in rats. This might be explained by a calcium-phosphate-induced increase in luminal buffering capacity, which protects against an acidic pH due to microbial fermentation. Therefore, we investigated whether dietary phosphate is a co-player in the effect of Ca on permeability. Rats were fed a humanised low-Ca diet, or a similar diet supplemented with Ca and containing either high, medium or low phosphate concentrations. Chromium-EDTA was added as an inert dietary intestinal permeability marker. After dietary adaptation, short-chain fructo-oligosaccharides (scFOS) were added to all diets to stimulate fermentation, acidify the colonic contents and induce an increase in permeability. Dietary Ca prevented the scFOS-induced increase in intestinal permeability in rats fed medium- and high-phosphate diets but not in those fed the low-phosphate diet. This was associated with higher faecal water cytotoxicity and higher caecal lactate levels in the latter group. Moreover, food intake and body weight during scFOS supplementation were adversely affected by the low-phosphate diet. Importantly, luminal buffering capacity was higher in rats fed the medium- and high-phosphate diets compared with those fed the low-phosphate diet. The protective effect of dietary Ca on intestinal permeability is impaired if dietary phosphate is low. This is associated with a calcium phosphate-induced increase in luminal buffering capacity. Dragging phosphate into the colon and thereby increasing the colonic phosphate concentration is at least part of the mechanism behind the protective effect of Ca on intestinal permeability

Intestinally secreted C-type lectin Reg3b attenuates salmonellosis but not listeriosis in mice

The Reg3 protein family, including the human member designated pancreatitis-associated protein (PAP), consists of secreted proteins that contain a C-type lectin domain involved in carbohydrate binding. They are expressed by intestinal epithelial cells. Colonization of germ-free mice and intestinal infection with pathogens increase the expression of Reg3g and Reg3b in the murine ileum. Reg3g is directly bactericidal for Gram-positive bacteria, but the exact role of Reg3b in bacterial infections is unknown. To investigate the possible protective role of Reg3b in intestinal infection, Reg3b knockout (Reg3b-/-) mice and wild-type (WT) mice were orally infected with Gram-negative Salmonella enteritidis or Gram-positive Listeria monocytogenes. At day 2 after oral Listeria infection and at day 4 after oral Salmonella infection, mice were sacrificed to collect intestinal and other tissues for pathogen quantification. Protein expression of Reg3b and Reg3g was determined in intestinal mucosal scrapings of infected and noninfected mice. In addition, ex vivo binding of ileal mucosal Reg3b to Listeria and Salmonella was investigated. Whereas recovery of Salmonella or Listeria from feces of Reg3b-/- mice did not differ from that from feces of WT mice, significantly higher numbers of viable Salmonella, but not Listeria, bacteria were recovered from the colon, mesenteric lymph nodes, spleen, and liver of the Reg3b-/- mice than from those of WT mice. Mucosal Reg3b binds to both bacterial pathogens and may interfere with their mode of action. Reg3b plays a protective role against intestinal translocation of the Gram-negative bacterium S. enteritidis in mice but not against the Gram-positive bacterium L. monocytogenes

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

Gene expression response of the rat small intestine following oral salmonella infection

Data on the molecular response of the intestine to the food-borne pathogen Salmonella are derived from in vitro studies, whereas in vivo data are lacking. We performed an oral S. enteritidis infection study in Wistar rats to obtain insight in the in vivo response in time. Expression profiles of ileal mucosa (IM) and Peyer's patches (PP) were generated using DNA microarrays at days 1, 3, and 6 postinfection. An overview of Salmonella-regulated processes was obtained and confirmed by quantitative real-time PCR on pooled and individual samples. Salmonella-induced gene expression responses in vivo are fewer and smaller than observed in vitro, and the response develops over a longer period of time. Few effects are seen at day 1 and mainly occur in IM, suggesting the mucosa as the primary site of invasion. Later, a bigger response is observed, especially in PP. Decreased expression of anti-microbial peptides genes (in IM at day 1) suggests inhibition of this process by Salmonella. Newly identified target processes are carbohydrate transport (increased expression in IM at day 1) and phase I and phase II detoxification (decreased expression at days 3 and 6). Increase of cytokine and chemokine expression occurs at later time points, both in PP and IM. Pancreatitis-associated protein, lipocalin 2, and calprotectin, potential inflammatory marker proteins, showed induced expression from day 3 onward. We conclude that the in vivo gene expression response of the ileum to Salmonella differs to a large extent from the response seen in vitro