Effect of dietary fats on endogenous formation of N-nitrosamines from nitrate in germ-free and conventional rats and rats harbouring a human flora.

Urinary excretion of N-nitrosoproline (NPRO) was measured in groups of four germ-free (GF) and conventional (CV) rats given a purified diet with or without inclusion of 100 g butterfat, coconut oil or maize oil/kg, and drinking water containing 0.235 M-NaNO3. In the CV environment rats given the fat-supplemented diets excreted significantly less NPRO than those on the low-fat diet. No corresponding decrease in NPRO excretion occurred in the GF environment. Nitrate reductase activity was measured in stomach contents and homogenates of stomach tissue from GF and CV rats given the different diets. No activity was detected in any of the contents from GF rats. Nitrate reductase activity was significantly reduced in contents from all the CV rats given the fat-supplemented diets, the effect being most marked in those given butterfat. Activity was much lower in tissue homogenates from GF rats than in those from their CV counterparts, but was not affected by diet in either environment. Groups of four CV rats, or rats harbouring a human faecal flora (HF), were given the purified diet with or without addition of 100 g butterfat or maize oil/kg and drinking water containing 0.235 M-NaNO3. All groups given the fat-supplemented diet with the exception of the HF rats given butterfat. It is concluded that the reduced excretion of NPRO by rats given diets containing fat was mainly due to inhibition of microbial nitrate reductase activity in the foregut. The smaller effect of butterfat on the HF rats accords with earlier findings in human subjects.

Factors influencing the presence of faecal lactobacilli in early infancy.

The faecal flora of 46 preterm infants and 52 born at full term was studied at 10 days of age; 46 born at full term and 37 preterm infants were also studied at 30 days. Viable counts of coliforms, lactobacilli, and bifidobacteria were made; gas liquid chromatography was used to identify the anaerobes. Lactobacilli, but not bifidobacteria, were found in high counts in the stools of most of the infants born at full term by 30 days of age. The mode of delivery, but not the method of feeding, had a significant influence on early colonisation. A selective deficiency of lactobacilli compared with coliform organisms was found in preterm infants. Previous treatment with antibiotics and being nursed in an incubator were also significantly associated with a lower rate of early colonisation with lactobacilli. Our findings indicate that lactobacilli may be an important part of the normal stool flora in early infancy, and that modern methods of neonatal care are associated with delayed or deficient colonisation.

The use of rats associated with a human faecal flora as a model for studying the effects of diet on the human gut microflora.

The activities of four bacterial biotransformation enzymes (beta-glucosidase, beta-glucuronidase, nitrate reductase and nitroreductase) were measured in the caecal contents of conventional flora rats or germ-free rats contaminated with a mixed, human faecal flora and compared with activities present in a fresh human stool preparation. Both the conventional flora rats and the rats inoculated with a human flora exhibited an enzyme profile generally similar to that of human faeces, although the conventional rat flora exhibited negligible nitrate reductase activity. The enzyme profile remained essentially unaltered in both human flora preparations following supplementation of the diet with pectin, whereas the conventional rat flora responded to this plant cell wall carbohydrate with a significant increase in nitrate reductase activity. The results demonstrate that enzymic activities of the human faecal microflora can be simulated in rats associated with a mixed population of human intestinal bacteria.

The influence of the host on expression of intestinal microbial enzyme activities involved in metabolism of foreign compounds.

The activities of four enzymes (beta-glucuronidase, nitrate reductase and nitroreductase) in selected intestinal bacteria (Escherichia coli, Clostridium sp., Streptococcus sp., Bacteroides sp. and Lactobacillus salivarius) were measured after growth in vitro and in vivo. The five strains differed in their activities with Clostridium sp. being the most active for beta-glucosidase, beta-glucuronidase and nitroreductase, and E. coli the most active producer of nitrate reductase. Enzyme activity in vivo tended to be higher than in vitro but there were instances where the comparative activities were reversed.

A note on the effect of host specific fermented milk on the coliform population of the neonatal rat gut.

An adhering strain of Lactobacillus salivarius isolated from the intestine of a rat was used to ferment cow's milk fortified with whey protein and threonine. When the fermented milk was used to dose baby rats orally for the first three days of life, the numbers of coliform organisms in the gut decreased significantly.

The effect of yoghurt on some components of the gut microflora and on the metabolism of lactose in the rat.

Feeding yoghurt or base milk (from which the yoghurt was prepared by fermentation) to rats increased the counts of coliforms in the gut whereas the counts of lactobacilli were reduced by yoghurt but not by the base milk. Lactobacillus bulgaricus survived in the guts of gnotobiotic and conventional rats when yoghurt was fed continuously. Streptococcus thermophilus also survived in gnotobiotic rats but its ability to survive in conventional rats could not be examined. Both organisms failed to colonise the gut when a small inoculum of yoghurt was administered orally to germfree rats maintained on the stock diet. Streptococcus thermophilus but not Lact. bulgaricus grew in the rat diet when tested in vitro. Two enzyme systems (beta-galactosidase and lactase) were studied using, respectively, o-nitrophenyl-beta-D-galactopyranoside (ONPG) and lactose as the test substrates. Enzyme levels estimated with both substrates increased in the gut contents when rats were fed yoghurt but an increase was only found with ONPG in the intestinal mucosa fraction. The bacterial origin of all this increased activity is discussed. The other lactose-containing diets did not affect enzyme activity to the same degree. Feeding yoghurt changed the lactobacillus flora from one which was predominantly heterofermentative (Lact. reuteri ) to one which was predominantly homofermentative (Lact. salivarius).

Bile acid deconjugation and attachment of chicken gut bacteria: their possible role in growth depression.

Bacteria isolated from the chicken gut were tested for their ability to deconjugate bile acids and attach to chicken epithelial cells (crop squamous cells and duodenal brush borders). Clostridium perfringens, streptococci and some of the bifidobacteria and lactobacilli were able to deconjugate all 4 substrates whereas the bacteroides deconjugated only the taurine conjugates and the coliforms were completely inactive. None of the strains of Escherichia coli or streptococci attached to squamous cells, but the anaerogenic coliform, the strain of Klebsiella aerogenes and the lactobacilli did show attachment. Attachment to brush borders was obtained with the anaerogenic coliforms, K. aerogenes, 2 out of 5 of the lactobacilli, and 4 out of 9 of the streptococci, but none of the strains of E. coli.

Trace-nutrient-binding proteins in milk and the growth of bacteria in the gut of infant rabbits.

1. The influence of trace-nutrient-binding proteins on the growth of coliforms, streptococci and lactobacilli in the gastrointestinal tract was examined in neonatal rabbits delivered germ-free and dosed with an artificial flora (ESL), or born conventionally and dosed with ESL or rabbit faeces. 2. In the stomach and small intestine of both gnotobiotic and conventional animals the counts of coliforms were usually atypically high and those of streptococci were always low. In the colon the counts of coliforms and streptococci were high. Lactobacilli usually became established in the gut of the gnotobiotic animals but were not found in the conventional rabbits. 3. Sterilization (freeze-drying followed by gamma-irradiation) of the milk decreased its capacity to bind added iron by 45% and vitamin B12 by 30%. When compared with raw milk, feeding of radiation-sterilized milk did not affect the viable count of coliforms and streptococci in the gut of gnotobiotic animals. 4. Saturating the nutrient-feeding proteins in milk with Fe, folic acid and vitamin B12 had no effect on the numbers of coliforms, streptococci and lactobacilli recovered from the intestine.

The gut microflora and the uptake of glucose from the small intestine of the chick.

1. Chicks whose growth rate had been depressed either by a fully conventional flora or by association with a bile acid deconjugating strain of Streptococcus faecium and/or a filterable agent from chicken droppings showed no significant reduction in uptake of 3-0-methyl-alpha-D-glucopyranose compared with germ-free birds. 2. Association with a microflora increased the weight of the gut per unit length.

The antimicrobial activity of cationic proteins isolated from the cells in bulk milk samples.

Cationic proteins isolated from the cells in bulk milk samples were shown to inhibit the growth of two pathogenic strains of staphylococci and also Streptococcus agalactiae S 13. Polyacrylamide gel disk electrophoresis studies on these proteins revealed the presence of at least 9 components some of which had isoelectric pH's between 7.0 and 9.0. Trace amounts of the isolated protein had isoelectric pH's greater than 9.0. Staphylococci incubated with milk-cell cationic proteins absorbed the protein, thereby allowing the organism to be stained with the anionic dye Fast Green FCF. Protein-treated staphylococci in isotonic solutions autoagglutinated. This autoagglutination was more marked in hypo- and hypertonic solutions. Lysozyme was not demonstrated in the isolated protein fractions in assays involving incubation with Micrococcus lysodeikticus for 90 min. The antimicrobial activity of the cationic proteins isolated from the bulk milk samples was not destroyed after heating to temperatures up to 70 degrees C for 30 min., whereas at higher temperatures the activity diminished and was almost completely lost at 100 degrees C.