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*
Department of Flavour, Nutrition and Ingredients, NIZO Food Research, 6710 BA Ede, The Netherlands; and the
Wageningen Center for Food Sciences, 6700 AN, Wageningen, The Netherlands
2To whom correspondence should be addressed. E-mail: sprong{at}nizo.nl.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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KEY WORDS: • calcium • dietary fat • fatty acids • bile acids • infection • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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–3
). Amorphous CaPi, formed in the pH neutral environment of the small intestine from dietary calcium and phosphate binds and precipitates fatty acids (4
) and bile salts (5
). Due to the precipitation, the total fecal excretion of surfactants is increased, whereas the concentration of bile salts and fatty acid in the water phase of intestinal contents is drastically decreased in rats and humans (4
–8
). The water phase of feces is believed to contain the cytolytic surfactants, whereas the precipitated bile salts and fatty acids of the insoluble phase are considered inert (4
). Salmonella, like most other Gram-negative bacteria, is insensitive to the lytic actions of bile salts and fatty acids (3
,9
,10
). In contrast, Gram-positive bacteria, including lactobacilli, are vulnerable to the bactericidal effect of these surfactants (3
,9
,10
). This is because Gram-positive bacteria lack the protective lipopolysaccharide (LPS)-rich outer membrane of Gram-negative bacteria (11
). Thus, CaPi, by precipitating noxious substances, may create a less cytotoxic intestinal environment that favors survival and growth of Gram-positive bacteria at the expense of their Gram-negative counterparts. Indeed, it has been shown that the CaPi-mediated reduction of S. enteritidis colonization coincides with a trophic effect on autochthonous ileal and fecal lactobacilli in rats (3
).
Endogenous commensal Gram-positive bacteria may not be the only ones that benefit from precipitation of bactericidal agents by dietary CaPi. Gram-positive pathogens may also take advantage of a less harsh intestinal environment. Therefore, we tested the hypothesis that high CaPi intake enhances infections caused by Gram-positive bacteria in a strictly controlled rat experiment. Rats were fed humanized diets containing low or high amounts of CaPi. Dietary fat consisted of corn oil or milk fat, two frequently consumed fats in Western societies. Corn oil is a rich source of 18:1 and 18:2, whereas milk fat contains high amounts of saturated fatty acids including 10:0, 12:0 and 14:0 (Table 1)
. 18:1, 18:2, 10:0, 12:0 and 14:0 were shown to be bactericidal for the Gram-positive bacterium Listeria monocytogenes (12
,13
). Precipitation of fatty acids by calcium is enhanced with saturated fatty acids (14
). We found earlier that dietary CaPi increases fecal excretion of fatty acids and bile salts and drastically lowers the concentration of these bactericidal surfactants in fecal water phase of rats fed diets containing either corn oil or milk fat (4
,7
). Therefore, we hypothesized that dietary CaPi promotes infection caused by Gram-positive bacteria because it lowers the bioavailability of the different bactericidal surfactants in rats fed corn oil or milk fat. Rats were orally inoculated with L. monocytogenes, a Gram-positive food-borne pathogen that colonizes the intestinal tract and translocates across both ileal and colonic epithelium (15
,16
), resulting in a systemic infection. Colonization was determined as well as translocation and induction of diarrhea.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Stock solutions of L. monocytogenes 4B (NIZO B1242, from the collection of our institute) stored at -80°C in brain heart infusion broth (BHI)3
(Difco, Detroit, MI) containing 20% (v/v) glycerol were thawed and 20 µL of this suspension was plated on PALCAM-Listeria selective agar (Merck, Darmstadt, Germany). Plates were aerobically incubated at 37°C for 18 h. Cells were inoculated from these plates into 5 mL BHI and grown overnight under aerobic conditions at 37°C. The next day, bacteria were collected by centrifugation (20 min, 4000 x g), washed twice in saline, and suspended in saline to a concentration of 
1012 colony forming units (CFU)/L. Chenodeoxycholic acid, deoxycholic acid (both obtained from Sigma, St. Louis, MO), and C12:0 (Fluka, Buchs, Switzerland) were dissolved in a buffer containing 50 mmol/L 3-N-morpholinopropanesulphonic acid (MOPS; Sigma) and 100 mmol/L NaCl, pH 7.0. Chenodeoxycholate was mixed with deoxycholate in a molar ratio of 2:1. Final concentrations of this bile acid mixture and of lauric acid ranged from 0 to 2 mmol/L. Bacteria (100 µL of the suspension mentioned above) were added to 5 mL of surfactant solutions. After an aerobic incubation for 2 h at 37°C, viable L. monocytogenes were enumerated by plating dilutions on PALCAM. Plates were aerobically cultured for 36 h at 37°C. The experiment was performed in triplicate.
Animals, diets and infection.
The experimental protocol was approved by the animal welfare officer of the Wageningen University, The Netherlands. Male Wistar rats (specific pathogen-free, WU; Harlan, Zeist, The Netherlands), 9 wk old with a body weight of 250 g, were individually housed (n = 8 per diet) in metabolic cages in a room with controlled temperature (22–24°C), relative humidity (50–60%), and dark/light cycle (light, 0600–1800 h). Rats were fed standard rat diets (Hope Farms, Woerden, The Netherlands) until they received test diets. Purified diets varying in amount of calcium phosphate (low CaPi, 20 µmol/g; high CaPi, 160 µmol/g) and type of dietary fat (Table 1)
were supplied as a porridge with 680 g dry weight/kg food (dry diets mixed with double-distilled water) during the entire experimental period of 17 d. After preparation of the diets, the calcium content was checked in dry ashed, HCl-acidified diet samples using an atomic absorption spectrophotometer (Model 1100; Perkin Elmer Corp., Norwalk, CT). Rats had free access to food and demineralized drinking water. Food intake and body weight were recorded every 3 d pre-infection and daily postinfection.
After 2 wk of habituation to diets and housing conditions, rats were deprived of food for 14 h, fed for 2 h, and subsequently orally inoculated with 1 mL saline containing 4 x 109 L. monocytogenes 4B (determined by plating on PALCAM). L. monocytogenes was cultured as described in the in vitro experiment. Before administration, L. monocytogenes was routinely orally passaged in two rats and isolated from spleen and liver to ensure invasiveness of the strain. Fecal excretion of L. monocytogenes was measured in fresh feces samples collected before infection and on d 1, 2 and 3 after inoculation. Feces were homogenized in 1 mL saline. 10-fold dilutions were plated on PALCAM, followed by incubation at 37°C for 36 h.
To study whether dietary fat affects the systemic capacity of phagocytes to produce oxidation products of nitric oxide (NOx) upon stimulation, rats (n = 6 per group) were fed low calcium diets containing the two different types of fat. After 2 wk of habituation to diets and metabolic cages, rats were intraperitoneally challenged with a single dose of LPS (1 mg/kg body weight suspended in 1 mL saline) derived from S. enteritidis (Sigma).
Urinary NO metabolites.
Urine (complete 24-h samples) was collected daily, starting 2 d before inoculation of L. monocytogenes or injection with LPS, until the end of the experiment. Oxytetracycline (Sigma; 100 times the minimal inhibitory concentration) was added to the urine collection tubes to prevent bacterial growth. Urinary NOx was measured using Griess reagent as described before (17
). Data from the infection experiment are expressed as the total Listeria-induced increase in NOx during 3 d as calculated by (
total NOx i) - (3 x NOxb); NOx i reflects the excretion of NOx after L. monocytogenes inoculation and NOxb the mean daily NOx excretion before infection (baseline excretion). Results from the LPS experiment are expressed as µmol NOx/d.
Fecal analysis.
Complete 24-h feces samples were collected during 3 d preinfection and 3 d postinfection. Feces were lyophilized for dry weight determination.
Diarrhea was determined by calculating the water content of feces using measurement of cations, as described before (18
). The fecal cations ammonia, potassium and sodium were measured as described previously (3
).
Reconstituted fecal water was prepared from lyophilized feces based on the calculated fecal wet weights. Because no fecal water can be obtained from samples with a wet weight percentage of <700 g/kg, the group with the lowest mean wet weight percentage was set at 700 g/kg (WWPl). The actual percentages of wet weight (WWPa) of other groups were corrected for this percentage using the formula: (WWPa/WWPl) x 700 = WWPfw. WWPfw reflects the percentage wet weight used for fecal water preparation. Fecal water was prepared as described before (19
).
Calcium was measured in dry ashed (8 h at 550°C), lyophilized feces after destruction (15 min at 180°C) with a mixture of 11.6 mol/L perchloric acid and 9.8 mol/L hydrogen peroxide (5:1 v/v). For the measurement of calcium in fecal water, trichloroacetic acid extracts (50 g/L trichloroacetic acid) were made. Calcium was measured using atomic absorption spectrometry. Recovery of a standard (CaCl2.2H2O) ranged from 94 to 107%. Inorganic phosphate was determined spectrophotometrically in trichloroacetic acid (50 g/L)-extracted freeze-dried feces (16
) and fecal water by the method of Fiske and Subbarow (20
). Recoveries of an added standard (K2HPO4) were between 93 and 115%.
Free fatty acids and bile acids were measured in extracts of fecal water and feces. For free fatty acids and bile acids in fecal water, samples (50 µL) were acidified with HCl (final concentration 1.25 mol/L) and subsequently extracted with 2.5 mL diethyl ether followed by centrifuging at 3500 x g for 5 min. Supernatants were aspirated and the extraction was repeated twice. Aspirates were pooled and evaporated and subsequently redissolved in ethanol. To measure total free fatty acids in feces, samples were acidified with HCl (final concentration 1 mol/L), and sonicated for 10 min followed by extraction with 3 x 2.5 mL diethyl ether. Pooled aspirates were dried under nitrogen and resuspended in ethanol. Total free fatty acids were assayed using an enzymatic method (NEFA-C kit; Wako Chemicals, Neuss, Germany). Recovery of standards (mixture of 12:0 and 16:0) was 102–109% and 86–99% for fecal water and feces, respectively. Bile acids were measured in feces using an enzymatic assay (Sterognost 3
-Flu; Nycomed AS, Norway) as described before (4
). Recovery of a standard (deoxycholate) ranged from 92 to 104% for fecal water and from 90 to 102% for feces samples.
Growth of L. monocytogenes in fecal extracts ex vivo.
Bacteria were cultured as described above. Fecal extracts (100 g/L) were made by incubating freeze-dried feces (collected preinfection) with saline at 37°C for 30 min. Extracts were centrifuged for 2 min at 14000 x g. Supernatants were inoculated with L. monocytogenes (final concentration 5 x 108 CFU/L), and incubated at 37°C under aerobic conditions. 10-fold dilutions of samples taken from the incubates at t = 0, 2, 4 and 6 h were cultured on PALCAM plates as described above.
Statistics.
Results from the in vitro experiment are presented as mean ± SD (n = 3). Data of in vivo experiments are given as mean ± SEM (n = 8). Two rats with rattling breathing were omitted from the high calcium corn oil group because their lungs were infected by L. monocytogenes due to reflux of intragastrically administered bacteria. Data were checked for homogeneity of variances and normality using Levene’s test and Normal Probability Plots, respectively. Differences between treatment groups were tested with the nonparametric Kruskal-Wallis ANOVA, followed by Mann-Whitney U test with Bonferroni’s correction for multiple comparisons. Statistics were performed with a commercially available statistical package (Statitistica 6.0; StatSoft, Inc., Tulsa, OK).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The mixture of bile acids dose-dependently decreased L. monocytogenes survival (Fig. 1
). Comparable results were obtained with lauric acid.
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Body weight gain did not differ between diet groups and was not affected by infection. Body weight was 253 g at the start of the experiment. Final body weight was 330 g. Dietary CaPi did not affect food intake (mean: 17.4 g/d). Infection with L. monocytogenes slightly decreased food intake equally in all diet groups (mean: 15.8 g/d).
Intestinal colonization, translocation and diarrhea.
Supplemental CaPi increased fecal excretion of L. monocytogenes in the corn oil group (Fig. 2
A), indicating that the intestinal survival and/or colonization of L. monocytogenes was stimulated 10-fold in this group. In contrast, fecal L. monocytogenes excretion was unaffected by CaPi intake in rats fed milk fat diets (Fig. 2
B). Except for d 2, fecal L. monocytogenes excretion differed (P < 0.05) among diet groups. This lack of difference on d 2 was due to the large variation in means of the high CaPi milk fat group.
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shows the Listeria-mediated increase in total urinary NOx corrected for basal NOx. Supplemental CaPi significantly augmented the Listeria-induced increase in NOx excretion in corn oil fed rats, indicating increased translocation. In contrast, dietary CaPi did not affect this variable in the milk fat group. The type of dietary fat did not affect the capacity of the inducible NO-synthase to respond to bacterial stimuli, because no difference in urinary NOx excretion was observed when rats, fed either corn oil or milk fat diets, where intraperitoneally injected with LPS (129 ± 23 and 102 ± 13 µmol/d 24 h after LPS-injection for corn oil and milk fat groups, respectively).
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High CaPi intake increased total fecal output in rats fed both types of dietary fat (Table 2)
. As expected, fecal calcium excretion was increased by high CaPi intake in rats fed both corn oil and milk fat diets. Concomitantly, fecal inorganic phosphate excretion was increased in these groups due to the intestinal formation of insoluble CaPi. The amount of fecal bile salts was not affected by supplemental CaPi in rats fed both types of dietary fat. CaPi supplementation significantly increased the amount of free fatty acids in feces in rats fed both types of dietary fat.
Composition of fecal water.
The pH of fecal water was not affected by CaPi intake (Table 2)
. The concentration of calcium in fecal water was significantly increased by supplemental CaPi in rats fed both types of diet. High CaPi intake did not affect the phosphate concentration. The concentration of bile salts was considerably decreased by high CaPi consumption, independent of the type of dietary fat. The concentration of bile salts in the high CaPi-corn oil group was significantly lower than those in the high CaPi- milk fat group. In addition, CaPi supplementation significantly decreased the concentration of free fatty acids in fecal water of rats fed corn oil diets. However, high CaPi intake did not affect the free fatty acid concentration in fecal water of rats fed milk fat diets.
Growth of L. monocytogenes in fecal extracts.
To mimic the capacity of L. monocytogenes to expand in the intestinal lumen, growth of this pathogen was measured in fecal extracts ex vivo. Except for at 6 h, growth of L. monocytogenes differed between groups (P < 0.05). Growth of L. monocytogenes was enhanced in fecal extracts of rats fed the high CaPi corn oil diet compared with the low CaPi corn oil diet (Fig. 4
A), but dietary CaPi did not affect L. monocytogenes growth in fecal extracts of rats fed milk fat diets (Fig. 4
B).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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(12
,13
). The concentrations of surfactants used in the in vitro study are physiologically relevant because comparable concentrations were determined in ileal contents (3
) and fecal water (Table 2)
(2
). In vivo, high CaPi intake indeed aggravated L. monocytogenes colonization in corn oil-fed rats (Fig. 2
A).
Because the systemic load of L. monocytogenes is directly proportional to the oral inoculum (21
) and luminal amount of the pathogen (16
), the CaPi-mediated increase in Listeria colonization may result in an increased translocation of the pathogen. High CaPi intake indeed promoted L. monocytogenes translocation as was measured by an increased urinary NOx excretion (Fig. 3)
. Our group has previously shown that urinary NOx, which reflects the production of NO, is a quantitative biomarker of translocation of S. enteritidis (17
) and L. monocytogenes (22
) across the intestinal epithelium. In contrast to microbiological culturing of lymphoid organs, which only measures viable bacteria and not the ones already killed by immune cells, measurement of urinary NOx assesses the total bacterial load. Our study showed that the capacity of NO production in LPS-injected rats is not influenced by the type of dietary fat. In addition, it has been shown that NO production is unaffected by dietary CaPi (2
). Thus, the excretion of urinary NO metabolites is a reliable and accurate variable to quantify translocation of L. monocytogenes. Therefore, the increased excretion of NOx in the high calcium corn oil group indicates that L. monocytogenes translocation is increased in this group.
In some cases L. monocytogenes enteritis in humans is accompanied by diarrhea (23
,24
). Therefore, diarrhea was determined by the Listeria-induced increase in water content of feces. Diarrhea was also significantly increased by high CaPi intake in rats fed corn oil diets (Table 2)
.
The results obtained with the corn oil-fed rats support our hypothesis. However, when dietary fat was provided as milk fat instead of corn oil, different results were obtained. Diarrhea, L. monocytogenes colonization and translocation were not affected by CaPi intake in rats fed milk fat diets. To gain further insight into this striking discrepancy, ex vivo growth of L. monocytogenes was measured in fecal extracts. In agreement, growth of Listerial cells was enhanced by high CaPi intake in the corn oil group but not in rats fed milk fat diets (Fig. 4)
. These results suggest that high dietary CaPi intake increases a growth-stimulating component or suppresses an inhibitory luminal factor in rats fed corn oil diets.
Because L. monocytogenes is susceptible to the lytic action of bile acids and fatty acids, fat-mediated differences in the intestinal interactions between CaPi and these listericidal surfactants may be responsible for the observed phenomenon. Although CaPi decreased soluble bile acids in rats fed both types of diets, the concentration of listericidal bile salts was significantly lower in rats fed the high CaPi corn oil diet compared with those fed the high CaPi milk fat diet (Table 2)
. Although total fatty acids were precipitated by CaPi in rats fed both types of dietary fat, high CaPi resulted in lower concentrations of soluble listericidal fatty acids in rats fed corn oil diets but not in those fed milk fat diets. Thus, the residual soluble bile salts and fatty acids may account for the observed differences in L. monocytogenes colonization. However, other unknown factors may also be involved. For example, the concentration of calcium in fecal water was much higher in the high CaPi, corn oil group compared with the high CaPi, milk fat group (Table 2)
. Because calcium is an important stabilizing agent of bacterial cell walls (25
), L. monocytogenes may benefit from this higher calcium concentration by increased membrane stability. Additional research is required to elucidate the precise interaction between CaPi intake, the type of dietary fat, and L. monocytogenes infection.
Based on our previous studies with Salmonella (1
–3
), supplemental CaPi seemed to be a promising tool to reduce the incidence of gut infections. This study, however, indicates that the interactions between dietary CaPi and food-borne pathogens are more subtle. Whereas the studies with S. enteritidis show that CaPi inhibits the infection with a Gram-negative pathogen, these results indicate that CaPi may increase infection caused by a Gram-positive pathogen. Thus, properties of the cell membrane of the bacterial pathogen in addition to the type of dietary fat may determine whether high CaPi intake turns out to be protective.
In conclusion, this study shows that dietary calcium enhanced the colonization of L. monocytogenes in the gastrointestinal tract, promoted the translocation of this pathogen to extra-intestinal tissues and increased diarrhea in corn oil-fed rats but not in rats fed diets containing milk fat. The mechanism of this different effect of dietary CaPi is at present not known and requires further investigation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: BHI, brain heart infusion; CaPi, calcium phosphate; CFU, colony forming units; LPS, lipopolysaccharide; NO, nitric oxide; NOx, nitrate and nitrite; WWPa, actual percentage wet weight; WWPl, lowest mean percentage wet weight; WWPfw, wet weight percentage used for fecal water preparation.
Manuscript received 3 December 2001. Initial review completed 4 January 2002. Revision accepted 26 February 2002.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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