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Articles

High Intake of Milk Fat Inhibits Intestinal Colonization of Listeria but Not of Salmonella in Rats¹

Section of Nutrition and Health, NIZO Food Research, 6710 BA Ede, The Netherlands

²To whom correspondence should be addressed.

	ABSTRACT

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During fat digestion, fatty acids and monoglycerides are liberated in the gastrointestinal tract. Generally, these lipids are potent inhibitors of gram-positive bacteria in vitro but have less effect on gram-negative microbes. Considering this, we hypothesized that increased intake of bovine milk fat would result in enhanced gastrointestinal killing of Listeria monocytogenes (gram-positive) but have little effect on infection with Salmonella enteritidis (gram-negative) in rats. To test this, rats were fed either low milk fat diets (10% of energy obtained from milk fat, corresponding to 4.2 g fat/100 g diet) or high milk fat diets (40% of energy obtained from milk fat, corresponding to 19.6 g fat/100 g diet). After adaptation to these diets, rats were orally infected with Listeria or Salmonella. Greater milk fat consumption in Listeria-infected rats diminished intestinal colonization of Listeria (P < 0.05) and reduced diarrhea (P < 0.05). Analysis of gastrointestinal contents showed that killing of Listeria occurred predominantly in the stomach. High milk fat intake significantly augmented this gastric listericidal capacity (P < 0.05) and raised the concentration of medium-chain and saturated long-chain free fatty acids and of monoglycerides of C12:0, C14:0, C16:0, C18:0, and C18:1 in gastric chyme (P < 0.05). Considering the in vitro listericidal capacity of these agents, it was concluded that particularly the free fatty acids C10:0, C12:0 and C14:0 and the monoglycerides of C12:0, C14:0, and C16:0 seem to play a pivotal role in this enhanced Listeria killing. In contrast, Salmonella infection was not affected by milk fat consumption. In conclusion, high milk fat intake results in higher concentrations of gastric bactericidal lipids and thereby protects against Listeria infection but not against Salmonella.

KEY WORDS: • fatty acids • bactericidal • gastrointestinal • infection • rats

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Gastrointestinal infections provoked by food-borne bacteria are an enormous problem for public health. Newborns, the elderly and immunocompromised subjects are particularly at risk. Due to the increasing resistance of pathogens to antibiotics, efforts to enhance the host's resistance to pathogens by a nutritional approach deserve attention. The host immune system consists of constitutive nonspecific defenses and inducible specific antibody-mediated defenses. Because full expression of specific defenses takes at least 1 wk, supporting nonspecific defenses could be successful in fighting intestinal infections. Luminal factors such as gastric acidity, antimicrobial bile salts and pancreatic enzymes, together with intestinal motility, epithelial mucin secretion, exfoliation of epithelial cells and autochthonous microflora contribute to intestinal nonspecific defenses by killing pathogens and preventing their colonization. We hypothesize that changing the composition of the diet and thus changing the composition of the gastrointestinal contents may affect survival and colonization of pathogens. For example, whole milk consumption in children is associated with fewer gastrointestinal infections than is consumption of low fat milk (Koopman et al. 1984 ). This may be attributed to the antimicrobial activity of milk lipids towards bacteria and viruses that has been observed in vitro (Isaacs et al. 1986, 1992 and 1995 ). Triglycerides in milk fat are not toxic in themselves but become active upon treatment with lipase (Isaacs et al. 1995 ), implying involvement of free fatty acids and partially hydrolyzed glycerides. Unlike diglycerides, fatty acids and monoglycerides are powerful antimicrobial agents in vitro (Conley and Kabara 1973 , Hentgens et al. 1995 , Isaacs et al. 1995 , Kabara et al. 1972 , Sheu and Freese 1973 ). The bactericidal effects of free fatty acids and monoglycerides depend on properties of the bacterial cell wall. Although some exceptions have been described, gram-positive bacteria are more sensitive than gram-negative bacteria (Conley and Kabara 1973 , Isaacs et al. 1995 , Kabara et al. 1972 , Sheu and Freese 1973 ). This is because the lipopolysaccharide-rich outer membrane protects gram-negative bacteria against cytotoxic surfactants (Sheu and Freese 1973 ).

Despite the well-known in vitro bactericidal effects, evidence of lipid-mediated protection against gastrointestinal infections in vivo is scarce. Because fat digestion yields fatty acids and monoglycerides, protection against microbes mediated by lipolytic products is a likely phenomenon in vivo. In this study, we further explored this concept by testing the hypothesis that high milk fat intake enhances resistance to the gram-positive Listeria monocytogenes by increasing gastrointestinal concentrations of fatty acids and monoglycerides, but will have less effect on the gram-negative Salmonella enteritidis. These pathogens were chosen for study because they are important food-borne pathogens in humans. First, sensitivity of these pathogens to lipolytic products was tested in vitro. Next, the effect of high milk fat intake was tested in a strictly controlled rat experiment. Colonization of pathogens was determined, as well as the bactericidal capacity and concentrations of fatty acids and monoglycerides of gastrointestinal contents.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Bacterial culturing.

Listeria monocytogenes 4B (clinical isolate) and Salmonella enteritidis phage type 1 (clinical isolate) were routinely stored at -80°C in brain heart infusion broth (BHI,³ Difco, Detroit, MI) containing 20% (v/v) glycerol. Stock suspensions were quickly thawed and subsequently plated on PALCAM (Merck, Darmstadt, Germany) and Brilliant Green Agar plates (BGA, Oxoid, Basingstoke, UK), for Listeria and Salmonella, respectively. Plates were aerobically incubated at 37°C for 18 h. Subsequently, a few colonies from appropriate agar plates were inoculated in BHI followed by overnight incubation at 37°C under aerobic conditions. Bacterial cells were collected by centrifugation (15 min at 3500 x g), washed three times in saline and resuspended in saline to prepare stock suspensions.

In vitro killing capacity of fatty acids and monoglycerides.

The sensitivity of Listeria and Salmonella to the lytic action of fatty acids and monoglycerides was tested in vitro. First, the killing capacity of a mixture of fatty acids, typical for milk, was tested. Fatty acids (C4:0, C6:0, C8:0, C10:0, C12:0 and C14:0, all obtained from Fluka, Buchs, Switzerland) were dissolved at a concentration of 20 mmol/L in 100 mmol/L KOH, and diluted in citrate-buffer pH 5.0 (100 mmol/L potassium citrate) to mimic the gastric environment. Fatty acids were mixed in the ratio 10:4:2:3:2:2 for C4:0, C6:0, C8:0, C10:0, C12:0 and C14:0, respectively, which is the molar ratio of these fatty acids in milk fat (Jensen and Newburg 1995 ). Final concentrations of the mixture were in the range of 0–2 mmol/L. Bacteria were cultured as described above. Approximately 10⁸ colony forming units (cfu) in 200 µL saline were inoculated into 800 µL of citrate buffer (pH 5.0) containing fatty acids. Samples were incubated in a shaking water bath at 37°C for 2 h. To test the listericidal effect of individual fatty acids, the above-mentioned fatty acids as well as C16:0, C18:0, C18:1 and C18:2 (all obtained from Fluka) were dissolved, diluted and incubated with Listeria as described above for the milk fat mixture. To determine the bactericidal capacity of individual monoglycerides, stock solutions of 50 mmol/L were prepared by dissolving the monoacyl glycerols C10:0, C12:0, C14:0, C16:0, C18:0, C18:1 and C18:2 (all obtained from Sigma, St. Louis, MO) in absolute ethanol. Stock solutions were diluted in citrate buffer (pH 5.0). Final concentrations ranged between 0 and 1 mmol/L. Control incubates contained 2% ethanol (v/v). Listeria and Salmonella were incubated with monoglycerides as described above. Immediately after inoculation with bacteria and after 2 h of incubation at 37°C, a sample was drawn and 10-fold dilutions were made in saline. Viable Listeria were enumerated by plating 10-fold dilutions on PALCAM followed by aerobic culturing at 37°C for 36 h. Salmonellae were counted by plating dilutions on BGA. Plates were subsequently cultured aerobically at 37°C for 18 h. The whole experiment was performed in triplicate. The detection limit was 5 log₁₀ cfu/L incubation medium.

Animals and diets.

The experimental protocol was approved by the animal welfare officer of the Agricultural University, Wageningen, The Netherlands. Specific pathogen–free male Wistar rats (WU, Harlan, Zeist, The Netherlands), 9 wk old, and with a body weight of ~320 g, were housed individually in metabolic cages. Rats were fed standard rat diet (Hope Farms, Woerden, The Netherlands) until they received test diets. Temperature (22–24°C), relative humidity (50–60%), and the dark:light cycle (light, 0600–1800 h) were kept constant.

Experiment 1.

Rats (eight per group) were fed purified diets containing 10 and 40 energy % fat (corresponding to 4.2 and 19.6 g milk fat/100 g diet, respectively; Table 1 ). Free fatty acids and monoglycerides in diets were measured by gas chromatography (see below). Levels of free fatty acids were 0.32 and 1.25 µmol/g in the low fat and high fat diets, respectively, whereas neither diet contained monoglycerides. The calculated energy density of the diets was 15.51 and 18.62 kJ/g for the low fat and high fat diets, respectively. The nutrient density (ratio of vitamins, minerals, protein and cellulose to energy) was kept constant. Diets were supplied as a porridge with 68% dry weight (dry diets mixed with double distilled water) to minimize food dissipation in order to accurately measure food consumption and to avoid contamination of feces with food. Rats were given free access to food and demineralized water. Food intake was recorded every 2 d before infection and daily after infection. Body weight was measured every 4 d preinfection and daily after infection. After 2 wk of habituation of rats to housing conditions and diets, rats were deprived of food for 14 h and subsequently fed for 2 h. Immediately after the feeding period, rats were orally infected by gastric gavage with 1 mL saline containing 5 x 10⁹ cfu L. monocytogenes or with 2 x 10⁹ cfu S. enteritidis, as enumerated by plating on PALCAM and BGA, respectively. Bacteria were cultured as described above. The virulence of the strains used was sustained by routine oral passage in Wistar rats, followed by isolation from spleen and liver. Excretion of viable Listeria or Salmonella was measured in fresh feces samples collected on d 1 and 3 after infection. Samples were weighed and subsequently homogenized in 1 mL saline. Tenfold dilutions made in saline were plated on PALCAM for the enumeration of Listeria and on Modified Brilliant Green Agar (Oxoid) supplemented with sulfomandelate (Oxoid) for the determination of Salmonella. Plates were cultured aerobically at 37°C for 36 and 18 h for Listeria and Salmonella, respectively. Detection limits were 5 log₁₀ cfu/L saline.

Experiment 2.

To determine the listericidal capacity of the contents of both the stomach and small intestine and to assess the fatty acid profiles in these gastrointestinal segments in noninfected animals, rats (n = 4/diet group) were fed as described in Experiment 1. After 2 wk of habituation to diets and housing conditions, rats were subjected to the food-depriving and feeding procedure described above. Rats were killed immediately after the 2-h feeding period. Gastrointestinal contents were collected as described above.

Fecal analyses.

Feces were collected during 4 d before infection and 3 d after infection. Feces were lyophilized for dry weight determination. Measurement of diarrhea by direct measurement of the water content of feces by weighing feces before and after freeze drying underestimates the real water content because of evaporation of water during the 24-h collection period, especially in diarrheal states. Therefore, the water content was calculated using the fecal concentration of sodium, potassium and ammonium, assuming that these cations and their counter anions are the main electrolytes in feces, and that osmolarity of intestinal contents is always 300 mOsmol/L, even under the condition of diarrhea (Fine et al. 1993 ). Cations were determined as described previously (Bovee-Oudenhoven et al. 1997 ).

Fecal water based on 30% dry weight was reconstituted using lyophilized feces as described previously (Bovee-Oudenhoven et al. 1997 ).

Gastric and intestinal characteristics.

Samples were thawed, and the pH was measured at 37°C using a Methrom 632 pH meter (Applikon, Schiedam, The Netherlands). Bactericidal capacity was tested in the gastrointestinal contents of both Listeria-infected and noninfected rats by mixing 40 mg chyme with 40 µL saline, followed by incubation with 20 µL saline containing 10⁷ cfu Listeria at 37°C for 2 h in a shaking water bath. Fecal water was diluted 1:1 with saline; 80 µL of this diluted fecal water was incubated with 20 µL saline containing 10⁷ cfu Listeria. For gastric incubates, samples were drawn directly after inoculation of bacteria and every 30 min thereafter. For intestinal and fecal incubates, samples were taken at 0 and 2 h. Saline was used to assess spontaneous killing of Listeria. To determine viable Listeria, 10-fold dilutions were plated on PALCAM plates, which were subsequently cultured aerobically at 37°C for 36 h. The detection limit was 5 log₁₀ cfu/L.

Identification of free fatty acids and monoglycerides.

Because bactericidal activity of gastrointestinal contents of infected rats were not different from that of noninfected rats, patterns of fatty acids and monoglycerides were measured in noninfected rats. Free fatty acids in contents of stomach and small intestine were measured as described for milk (De Jong and Badings 1990 ) with a few modifications. Briefly, 100 mg of gastric chyme or 50 mg of intestinal contents was diluted in 500 µL double distilled water, 100 µL of 5 mol/L H₂SO₄ and 200 µL of an internal standard consisting of 1 mmol/L C7:0, C13:0, and C17:0 dissolved in ethanol (absolute). Samples were mixed in an ultrasonic bath for 10 min. Fatty acids were extracted with 3 x 1.5 mL diethyl ether/hexane (1:1, v/v). After centrifugation at 3500 x g for 5 min at 15°C, the upper solvent layer was transferred to a collection tube. Fatty acids were isolated according to Kaluzny et al. (1985) with a few modifications. Briefly, diethyl ether/hexane extracts were applied to hexane-preconditioned aminopropyl columns (100 mg, 1 mL; Isolute, International Sorbent Technology LTD, Mid Glamorgan, UK), followed by elution of neutral lipids with 2 x 0.5 mL chloroform/2-propanol (2:1, v/v). Fatty acids were eluted with 2 x 0.5 mL of diethyl ether containing 0.43 mol/L formic acid. Samples were analyzed using gas chromatography as described previously (De Jong and Badings 1990 ).

Monoglycerides in gastrointestinal contents were determined as follows. Chyme (250 mg) was mixed with 250 µL double distilled water and 200 µL absolute ethanol containing 2.5 mmol/L of the internal standard sn-1 C19:0 monoacyl glycerol. Samples were mixed in an ultrasonic bath for 5 min and subsequently extracted three times with 2.5 mL diethyl ether/heptane (1:1, v/v). Extracts were fractionated according to Kaluzny et al. (1985) with a few modifications. Briefly, extracts were applied to heptane-preconditioned aminopropyl columns. The passaged extract was stored, mixed with the chloroform/2-propanol (2:1 v/v; 2 x 0.5 mL) eluate and subsequently evaporated under nitrogen. The residue was solubilized in heptane and applied to another heptane-precondotioned aminopropyl column. Triglycerides were removed with 0.48 mol/L diethyl ether and 1.56 mol/L dichloromethane in heptane and diglycerides with 2.55 mol/L ethyl acetate in heptane; then monoglycerides were eluted with chloroform/methanol (2:1, v/v). The monoglyceride fraction was evaporated under nitrogen, and subsequently silylated and analyzed using a modification of the method described by Plank and Lorbeer (1992) . Samples were incubated with reagent containing N,O-bistrimethyl-silyltrifluoroacetamide, pyridine and trimethylchlorosilane (5:5:1, v/v/v; all obtained from Pierce, Rockford, IL) at 80°C for 30 min. Incubates were evaporated under nitrogen, solubilized in heptane and subsequently subjected to gas chromatography. Analyses were performed with a Carlo Erba Model Mega 5160 gas chromatograph (Carlo Erba, Milan, Italy) equipped with an automatic on-column injector (Carlo Elba AS-550), a flame-ionization detector and a silica capillary column 30 x 0.32 mm coated with DB1-HT (d_f = 0.1 µm; Chrompack, Middelburg, the Netherlands). Direct cold on-column injection took place at an oven temperature of 95°C. After an isothermal period of 1 min, the oven temperature was increased at a rate of 10°C/min to 180°C, which was held for 1 min, followed by a rate of 20°C/min to 370°C, which was held for 10 min. The carrier gas (hydrogen) flow rate was 6.4 mL/min. Standards of sn-1 monoacyl glycerols (C10:0, C12:0, C14:0, C16:0, C18:0, C18:1 and C18:2) and sn-2 C16:0 monoglyceride were used for determining retention times. sn-2 Monoglycerides appeared in chromatograms exclusively as sn-1 isomers as a result of redistribution of fatty acids during the isolation and derivatization procedure.

Statistics.

Results of in vitro experiments are expressed as means ± SD (n = 3). The in vitro studies are two-factor models with pathogen and fatty acid concentration or type of lipid and concentration as sources of variation. Two-way ANOVA was used to test the differences. The Student-Newman-Keuls test for multiple comparisons was performed to identify lipids that differ from each other. Results of the in vivo experiments are shown as means ± SEM, with n = 4 for noninfected rats and n = 8 for infected rats. The in vivo studies are single-factor models with the amount of fat as the source of variation. Therefore, differences between low fat and high fat groups were tested by Student's t test for independent samples (one-sided). When preinfection and postinfection measurements were performed, e.g., energy intake, growth or diarrhea markers, the infection-induced change, i.e., the difference between preinfection and postinfection, was subjected to statistical analysis. Two-way ANOVA was applied to test the difference in gastric listericidal capacity with amount of dietary fat and time as independent factors. The level of significance was preset at P < 0.05. Statistical tests were performed using SPSS/PC+ (version 2.0, SPSS, Chicago, IL).

	RESULTS

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Bactericidal effects of lipids.

The mixture of fatty acids typical for milk fat significantly killed L. monocytogenes in a dose-dependent manner (P < 0.001), whereas much less killing of Salmonella was observed (P < 0.001; Fig. 1A ). The listericidal capacity of individual fatty acids depended on concentration (P < 0.001) and chain length (P < 0.001), and ranked in the order C14:0 < C18:2 < C10:0 < C18:1 < C12:0 (Fig. 1 B). The long-chain fatty acids C16:0 and C18:0 were not listericidal. In addition, C4:0, C6:0, and C8:0 were not listeridicidal in this concentration range. Even concentrations of 10 mmol/L C4:0 and C6:0 did not kill Listeria (not shown). However, 5 mmol/L C8:0 completely killed Listeria (not shown). Listeria was also more sensitive to monoglycerides than Salmonella (Fig. 2 ), and killing of both pathogens depended on concentration (P < 0.001) and chain length (P < 0.001). The listericidal activity of monoacyl glycerols ranked in the order C18:1< C16:0 < C14:0 < C10:0 < C18:2 < C12:0. The monoacyl glycerol of C18:0 was not lytic at all. Salmonella was moderately susceptible to C10:0 monoglyceride and almost insensitive to the monoacyl glycerols of C12:0 and C14:0.

View larger version (19K): [in this window] [in a new window]   Figure 1. Bactericidal effects of a mixture of fatty acids typical for milk on Listeria monocytogenes and Salmonella enteritidis (panel A), and of individual fatty acids on Listeria (panel B) in vitro. Viability of pathogens was determined by plating techniques after an incubation period of 2 h at 37°C. Data represent means and SD (sometimes smaller than symbols) of triplicate incubations. Due to overlap, the individual symbols of C4:0, C6:0, C16:0 and C18:0 cannot be distinguished.

View larger version (20K): [in this window] [in a new window]   Figure 2. Bactericidal capacity of monoglycerides for Listeria monocytogenes (panel A) and Salmonella enteritidis (panel B) in vitro. The viability of these strains was determined by plating techniques after 2 h of incubation at 37°C. Data represent means ± SD (sometimes smaller than symbols) of triplicate incubations.

As might be expected from the energy density of the diets, food intake was significantly lower in high fat groups compared with low fat groups (Table 2 ). Energy intake, however, did not differ between diet groups (mean, 362 kJ/d). Food intake and therefore also energy intake declined after both Listeria and Salmonella infection, but the total infection-mediated decrease in energy intake, i.e., the difference between pre- and postinfection, did not differ between the diet groups (Listeria, 85 kJ/d; Salmonella, 54 kJ/d). Growth rates were not affected by either diet or infection (mean, 4.7 g/d). Mean body weights were 320 g at the start of the experiments. Final body weights in Listeria-infected rats were 377 ± 10 g for the low milk fat group and 390 ± 8 g for rats consuming high milk fat diets. Final body weights in Salmonella-treated rats were 377 ± 6 g and 386 ± 8 in low fat and high fat groups, respectively.

Consumption of the high milk fat diet significantly diminished fecal excretion of Listeria at d 1 and 3 postinfection (Table 3 ) compared with the low milk fat group. In contrast, fecal Salmonella excretion was not altered at d 1 and tended to be greater (P = 0.07) at d 3 in rats fed high milk fat diets. These results indicate that high consumption of milk fat diminishes the intestinal colonization of L. monocytogenes but not that of S. enteritidis.

Listericidal capacity and composition of gastrointestinal contents.

Gastric contents possessed an enormous Listeria-killing capacity in infected rats (Fig. 3 ) and noninfected rats (not shown). Killing was time dependent, i.e., almost complete killing was observed after 2 h of incubation in rats fed high milk fat diets. Bactericidal activity was enhanced with the amount of milk fat consumed. Because listericidal capacity did not differ in infected and noninfected rats, only data for luminal contents of noninfected rats are given. Gastric pH was not significantly influenced by the amount of dietary milk fat (5.05 ± 0.23 in rats fed low fat diets vs. 4.53 ± 0.23 in rats fed high fat diets). Butyric acid, medium-chain and long-chain fatty acids were observed in total gastric contents (Fig. 4A ). Except for unsaturated C18:0 fatty acids, larger concentrations of free fatty acids were observed when larger amounts of dietary fat were consumed. Small amounts of monoglycerides with chain length 14 carbon atoms were observed in rats fed low fat diets (Fig. 5A ). Except for C18:2 monoglyceride, higher concentrations of these monoglycerides and also C12:0 monoacyl glycerol were measured in rats fed the high fat diet.

View larger version (17K): [in this window] [in a new window]   Figure 3. Listericidal activity of gastric contents of rats fed low milk fat or high milk fat diets. Killing was measured by incubating saline (control) or saline-diluted chyme (1:1, wt/v) with ~1010 cfu/L Listeria monocytogenes at 37°C. Viable Listeria were determined by plating techniques. Data represent means ± SEM (n = 8). Listericidal capacity was time dependent (P < 0.01) and was greater in the high fat group (P < 0.001).

View larger version (24K): [in this window] [in a new window]   Figure 4. Luminal free fatty acids of stomach (panel A), proximal (panel B) and distal (panel C) small intestine of noninfected rats fed diets containing low milk fat or high milk fat. Data are expressed as means ± SEM (sometimes invisible), n = 4. * Significantly different from the low fat diet group, P < 0.05.

View larger version (18K): [in this window] [in a new window]   Figure 5. Monoglyceride patterns in lumen of stomach (panel A), proximal (panel B) and distal (panel C) small intestine of noninfected rats. Data represent means and SEM (sometimes invisible), n = 4. * Significantly different from the low fat diet group, P < 0.05.

No luminal listericidal capacity was observed in the distal small intestine. Listeria counts after 2 h of incubation with chyme were 11.14 ± 0.03 and 11.10 ± 0.03 log₁₀ cfu/L in rats fed low fat and high fat diets, respectively. Control saline incubations contained 10.76 ± 0.03 log₁₀ cfu/L. The luminal pH was not affected by the amount of fat intake (low fat, 6.94 ± 0.07; high fat, 6.88 ± 0.14). Fatty acids with a chain length < C12:0 were not detectable in this part of the gastrointestinal tract. The concentration of the remaining C12:0, C14:0, C16:0 and c18:0 was magnified in rats fed high fat diets (Fig. 4 C), whereas the amount of unsaturated C-18 fatty acids was not enhanced. This part of the small intestine contained few monoglycerides in rats fed low fat diets and only very small amounts of C14:0, C16:0, and C18:0 monoacyl glycerols in rats fed high fat diets (Fig. 5 C).

No killing was observed when Listeria was exposed to fecal water (not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
This study shows that high milk fat intake in rats is associated with an improved resistance toward Listeria monocytogenes compared with low milk fat consumption. Fecal excretion of Listeria was significantly lower in rats fed high milk fat diets compared with rats fed low fat diets (Table 4), indicating that intestinal colonization of Listeria was diminished. The protective effect of high milk fat intake is supported by an abated Listeria-induced diarrhea in this group, as determined by the water content of feces, which was calculated using excretion of fecal cations. In contrast, high milk fat intake did not affect infection provoked by Salmonella enteritidis because no effect on fecal excretion of Salmonella and diarrhea parameters was observed. These results indicate that the infection-reducing capacity of milk fat is pathogen dependent.

To reveal the location and mechanism of high fat–mediated reduction of Listeria colonization, the listericidal capacity of gastrointestinal contents was determined. Killing of Listeria occurred predominantly in gastric chyme and was enhanced in rats fed high fat diets (Fig. 2) . Except for a slight listericidal capacity of the contents of the proximal small intestine of rats fed high fat diets, no indication of luminal Listeria killing in either the small or large intestine was observed in this study. Therefore, lipid-mediated Listeria killing probably occurs in the gastric lumen. Because the acidic environment of the stomach forms the first line of defense against acid-sensitive pathogens such as L. monocytogenes (Salyers and Whitt 1994 ), changes in gastric acidity may affect survival of pathogens. In our study, gastric pH was not significantly affected by the amount of fat provided, indicating that the enhanced listericidal capacity of gastric contents and the decreased gastrointestinal colonization of Listeria in vivo in the high milk fat group cannot be explained by differences in gastric pH.

Because the ratio of vitamins, minerals, proteins and cellulose to energy was kept constant, and energy intake was not affected by diet, nutrient intake of rats differed only in dextrose and fat contents. Because Listeria utilize dextrose for growth (Welshimer 1981 ), it is possible that the higher dextrose consumption of rats fed low fat diets may enhance proliferation of Listeria. The main difference in Listeria survival, however, was observed in gastric contents. Because the Listeria strain used in our study is not able to grow at pH 5 (not shown), i.e., the pH of the gastric environment, it is not likely that the glucose content of the diet accounts for the observed difference in Listeria colonization. Thus, the amount of dietary fat is most likely responsible for the observed effects. It has been shown that lipid fractions of gastric aspirates obtained from human infants show bactericidal activity (Isaacs et al. 1990 ). Fat digestion starts in the stomach by preduodenal lipases, derived either from lingual glands as has been described for rats (Armand et al. 1990 ) or from the gastric mucosa as has been described for humans (Borel et al. 1991 ), and yields fatty acids and partly hydrolyzed glycerides (Armand et al. 1995 ). Monoglycerides and fatty acids, particularly those that are protonated, have been shown to be powerful listericidal agents in vitro (Wang and Johnson 1992 ). Moreover, fatty acids have been identified as bactericidal factors of rabbit gastric chyme (Canas-Roderiquez and Smith 1966 ). Provided that lipolytic activity is not saturated, high fat intake may increase concentrations of gastric lytic lipids by providing more lipase substrate. Because high fat–mediated induction of rat lingual lipase activity has been described (Armand et al. 1990 ), an increased release of gastric lipolytic products with bactericidal activity is a likely phenomenon in animals fed high fat diets. In our study, high milk fat intake considerably augmented gastric fatty acids and monoglycerides (Figs. 4 A and 5A). Considering the dose-dependency of the listericidal capacity of these lipolytic products (Figs. 1 A and Fig. 2 A), it is conceivable that the higher amounts of bactericidal lipids are responsible for the diminished colonization of Listeria in rats fed high milk fat diets. In addition to different concentrations of listericidal lipids, increasing the length of exposure to these agents may also contribute to the diminished colonization of Listeria in rats fed high fat diets. As shown in Figure 3 , Listeria killing by gastric contents was time dependent. In vivo, gastric emptying is delayed in humans (Akrabawi et al. 1996 ) and rats by increased amounts of fat consumed (Kalogeris et al. 1983 ). Therefore, high milk fat intake results in a prolonged gastric exposure of Listeria to higher concentrations of listericidal lipids.

This study also indicated which gastric lipolytic products are responsible for the enhanced killing of Listeria by high milk fat intake. Figure 4 A shows that unsaturated C-18 fatty acids are not augmented and may therefore not account for the enhanced listericidal capacity. In contrast, fatty acids with chain lengths varying from C4:0 to C18:0 were increased in gastric contents of rats fed high fat diets. Because Figure 1 shows that only C10:0, C12:0 and C14:0 fatty acids possessed listericidal activity, these fatty acids are likely candidates. It is generally accepted that C12:0 is extremely toxic for gram-positive bacteria (Kabara et al. 1972 , Sheu and Freese 1973 ), and lytic effects have already been described for Listeria (Wang and Johnson 1992 ). Although less active than C12:0, C10:0 and C14:0 are also toxic for gram-positive bacteria (Kabara et al. 1972 , Sheu and Freese 1973 ). According to Wang and Johnson (1992) , C14:0 is not toxic for Listeria. These authors, however, tested listericidal effects of C14:0 only at pH 6.0 and not at pH 5.0. In addition to fatty acids, the highly listericidal monoacyl glycerols of C12:0, C14:0 and the moderately lytic 16:0 monoglyceride (Fig. 2) are also increased in gastric chyme of rats fed high fat diets (Fig. 5 A). Bactericidal effects of C12:0 and C14:0 monoglycerides have also been described by others (Conley and Kabara 1973 , Kabara et al. 1972 ).

Despite high concentrations of free fatty acids in the proximal small intestine (Fig. 4 B), only a minor listericidal activity was observed in the high milk fat group. This cannot be ascribed to differences in fatty acid profiles because the concentration of highly and moderately listericidal fatty acids in the proximal small intestine of rats fed high milk fat diets is slightly reduced (C10:0), or similar (C12:0) or even augmented (C18:1) compared with gastric fatty acids (Fig. 4) . Protonation of free fatty acids increases their listericidal effects (Wang and Johnson 1992 ). Therefore, it is feasible that the lack of Listeria killing in the small intestine is due in part to the higher pH (small intestine, 5.8; stomach, 4.5–5). In addition, the concentration of the listericidal monoglycerides of C12:0, C14:0, C16:0 and C18:2 is considerably lower in the proximal small intestine compared with the stomach and may thus also be responsible for a reduced Listeria elimination.

The involvement of fatty acids and monoglycerides in the high milk fat–mediated decrease of intestinal colonization of L. monocytogenes is supported by the observation that infection induced by S. enteritidis was not affected by a greater milk fat intake. This is consistent with the in vitro observation that Salmonella is less vulnerable to the lytic effects of lipids (Figs. 1 and 2 , Kabara et al. 1972 , Petschow et al. 1998 , Sheu and Freese 1973 ), and with the observation that most gram-negative bacteria are not lipid-sensitive whereas most gram-positive are (Conley and Kabara 1973 , Isaacs et al. 1995 , Kabara et al. 1972 ). However, some exceptions have been described. For example, Vibrio cholerae (Petschow et al. 1998 ) and Helicobacter pylori (Petschow et al. 1996 ) can be killed by the monoacyl glycerol of C10:0. In this study, Salmonella was also moderately sensitive to C10:0 monoglyceride. The low gastric concentration of this agent may be insufficient for the killing of Salmonella.

Gastric fat digestion in humans is catalyzed by gastric lipases rather than by lingual lipases. However, the specificity of rat lingual lipases is comparable to that of human gastric lipases (Hamosh 1984 ). Like rat lingual lipases, the activity of human gastric lipases is increased by high fat consumption (Armand et al. 1995 ). In healthy humans, lipolysis catalyzed by gastric lipases reaches 10–20% (Cohen et al. 1971 ), whereas in neonates, 40–60% of dietary fat is digested in the stomach (Moreau et al. 1988 ). Bactericidal activity of the lipid fraction of gastric aspirates has indeed been shown in neonates (Isaacs et al. 1990 ). Therefore, it is likely that high milk fat intake may also protect against gastrointestinal infections caused by lipid-susceptible pathogens in humans. Neonates, which form a population at risk, may benefit particularly from high milk fat intake. Our results indicate that protection against lipid-sensitive pathogens may not be specific for milk fat. For instance, coconut oil is a rich source of C10:0, C12:0 and C14:0 (Passmore and Eastwood 1986 ) and may therefore also be protective. Further research is required to study whether other dietary fats are also protective in vivo.

In conclusion, this study provides in vivo evidence that the amount of milk fat intake modulates gastrointestinal infections. High milk fat consumption reduced the intestinal colonization of Listeria monocytogenes but did not change infection provoked by Salmonella enteritidis. Enhancing the listericidal capacity of the stomach by augmenting the concentrations of bactericidal fatty acids such as C10:0, C12:0 and C14:0, and of C12:0, C14:0 and C16:0 monoglycerides seems to be a crucial step in the high fat–mediated protection against Listeria infection.

	ACKNOWLEDGMENTS

 
We thank Rob Dekker for performing the analysis of free fatty acids and help with analysis of monoglycerides, Annelies Landman-Schouten for expert biotechnical assistance and Roald Neeter for kindly providing C19:0 monoglyceride.

	FOOTNOTES

 
¹ Supported in part by the Netherlands Ministry of Agriculture, Nature Management, and Fishery.

³ Abbreviations used: BGA, Brilliant Green Agar; BHI, brain heart infusion; cfu, colony forming units.

Manuscript received January 13, 1999. Initial review completed February 24, 1999. Revision accepted April 16, 1999.

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