Effect of a high-beef diet on the fecal bacterial flora of humans.

Ten human volunteers completed a 4-month diet series consisting of 1 month each of a control diet, a meatless diet, a high-beef diet, and the same control diet. Fat and fiber contents were essentially the same in all four diets, but protein content was doubled during the high-beef diet. During the 4th week on each diet, three stool specimens collected from each volunteer were analyzed for chemical composition and content of facultative, aerobic, and anaerobic bacteria. The bacteriological data are presented in this paper. High beef protein consumption had little effect on the composition of the intestinal flora. There were no significant differences in total counts of facultative and aerobic or anaerobic organisms in the feces when volunteers were on meatless or high-beef diets. At the species level, when counts during the two control diets were comparable, in only three instances did the change from the meatless to a high-beef diet significantly influence the bacterial numbers. The ratio of mean counts of anaerobic to facultative and aerobic organisms was approximately 15:1 during the meatless diet and 34:1 during the high-meat diet. The data indicate that animal protein consumption has little effect on the fecal bacterial profile in humans.

Fecal flora of volunteers on controlled diets.

Ten volunteers completed a 4-month diet series consisting of a control diet, a meatless diet, a high-beef diet, and the same control diet, each lasting 1 month. Fat and fiber contents were essentially the same in all four diets, but protein content was doubled during the high-beef diet. High-beef protein consumption had little effect on the composition of the intestinal flora of the volunteers. There were no significant differences detween the high-beef and meatless diets in total numbers of anaerobic, facultative, or aerobic organisms isolated, nor were there major differences in the counts of the individual genera and species of bacteria encountered. The data indicate that animal protein consumption has little effect on the fecal bacterial profile in humans. The possible influence of animal fat consumption on the metabolic activities of intestinal bacteria is discussed.

Soluble plasma antigen in experimental Salmonella typhimurium infection in mice.

To detect and characterize Salmonella antigen in blood, outbred CF-1 female mice were inoculated intraperitoneally with S. typhimurium LT-2 and blood was assayed by ELISA for Salmonella common structural antigen. Plasma antigen was detectable early in the course of infection and increased in quantity later in the course of illness when animals showed high grade bacteremia and high counts of splenic bacteria. Antigen was associated with a cell-free plasma fraction of blood, passed through filters with cut-offs of 0.2 mu and molecular mass of 1000 kDa, and was enhanced in detectability after heating to 100 degrees C for 15 min. Antigen was concentrated by diluting plasma 1:4 in 0.1 M EDTA, heating to 100 degrees C, and concentrating the supernate with an ultrafiltration membrane with a molecular mass cut-off of 15 kDa. By gel filtration, antigen was associated with a peak at about molecular mass 300 kDa in heated plasma and a peak at about 380 kDa in unheated plasma. These results indicate that murine typhoid infection results in circulating soluble plasma antigen, which is heat-stable with a molecular mass of approximately 300 kDa.

Effect of diet on fecal nutrients.

Calculated and chemically analyzed nutrient values for three weighed constant diets were dissimilar for calcium, magnesium, protein, and fat, and similar for fiber and ash. Fecal nutrient concentrations of ten healthy men fed the same constant diets were quite variable. Feeding constant weighed diets of similar composition does not assure the same fecal residue in the large colon of human subjects.

Chemotaxis by lipids isolated from Bacteroides fragilis.

Living, heat or formalin killed Bacteroides fragilis and a crude preparation of their cell walls were examined by the Boyden technique for chemotactic activity upon guinea pig peritoneal exudate cells. Their relative chemotactic activity ranged from 3.0 to 5.2 compared to an average value of 6.4 for the positive control, an endotoxic culture filtrate of Escherichia coli. A culture filtrate of B. fragilis and an index of 3.7. Miocrogram quantities of cytoplasmic preparations obtained by ammonium sulphate precipitation had chemotactic indices ranging from 2.8 to 6.4, the highest value being displayed by the precipitate formed between 50 and 75% saturation with ammonium sulphate. This fraction retained leucotactic activity after exposure to strong acid and heat. The leucotactic potency of these fractions did not correlate directly with their protein content. Further precipitation of the most active fraction with 80% ethanol revealed that there was little chemotactic activity attributable to polysaccharides. Gas liquid chromatography of a chloroform-methanol extract of the cells which had a chemotactic index of 6.1 revealed the presence of more than thirty fatty acids ranging in carbon length from C8 to C25. These results suggest a role of lipids as initiators of the leucotactic response associated with infections caused by B. fragilis.

The anaerobic microflora of the human body.

An overview is presented of the kinds of anaerobic bacteria that inhibit the surfaces of the human body. The anaerobic floras of the skin, oral cavity, alimentary tract, and genitourinary tract are described. The activities of these organisms that impact on the human host and their interactions are discussed. Emphasis is placed on the protective roles of the floras of the various bodily surfaces in preventing infections. Identified mechanisms of protection on the skin and in the colon and the vagina are explained.

Inhibition of Shigella flexneri by the normal intestinal flora. I. Mechanisms of inhibition by Klebsiella.

Growth curves were plotted for Shigella flexneri and Klebsiella (Aerobacter aerogenes) multiplying in pure and mixed culture. In mixed culture, Klebsiella inhibited Shigella. Exponential growth was interrupted and Shigella entered into a logarithmic death phase. An analysis of cultures at the time inhibition occurred revealed that formic and acetic acids produced by Klebsiella were responsible for the inhibition of Shigella. Klebsiella strongly reduced the culture medium. The volatile fatty acids, operating under reduced conditions, exerted a bactericidal effect on Shigella. Results are discussed with reference to the possible role of volatile fatty acids as factors responsible for Shigella inhibition in vivo.

Inhibition of Shigella flexneri by the normal intestinal flora. II. Mechanisms of inhibition by coliform organisms.

Of 15 strains of coliform bacteria, all isolated from human feces, 14 inhibited the growth of Shigella flexneri in mixed culture. In every case, when inhibition occurred, exponential growth of Shigella was interrupted in the mixed culture and the organisms entered into either a stationary or a death phase. None of the test coliform strains produced colicines active against Shigella. An analysis of mixed-culture environments at the time Shigella inhibition occurred revealed that the inhibition was not due to nutrient depletion nor to the development of adverse pH or oxidation-reduction potentials in themselves. In mixed cultures, the coliform strains produced formic and acetic acids in concentrations that inhibited Shigella growth. With one exception, the coliform strains also greatly reduced the culture medium. In average concentrations produced, the formic and acetic acids exerted a bactericidal effect on Shigella under the reduced conditions found in mixed cultures. The acids were only moderately toxic for the coliform strains under the same conditions. Results indicate that volatile acid production and concomitant reduction of the medium are the mechanisms by which coliform bacteria inhibit Shigella growth in mixed cultures.

The intestinal flora and infant botulism.

The intestinal flora of experimental animals interferes with infection by species of Salmonella and Shigella. Protection against infection with these organisms appears to be related to high concentrations of volatile acids, low pH, and low oxidation-reduction potential of the intestinal contents of animals with an intact flora. There are no data to show that the flora influences colonization of the intestine with clostridial species, but indirect evidence suggests that the intestinal flora may be involved in this process. The impact of the intestinal flora on the ecology of the large intestine may be the most important determinant of resistance to infant botulism.

Differential effects of oxygen and oxidation-reduction potential on the multiplication of three species of anaerobic intestinal bacteria.

The sensitivity of three strains of anaerobic intestinal bacteria, Clostridium perfringens, Bacteroides fragilis, and Peptococcus magnus, to the differential effects of oxygen and adverse oxidation-reduction potential was measured. The multiplication of the three organisms was inhibited in the presence of oxygen whether the medium was at a negative oxidation-reduction potential (Eh of -50 mV), poised by the intermittent addition of dithiothreitol, or at a positive oxidation-reduction potential (Eh of near +500 mV). However, when these organisms were cultured in the presence of oxygen, no inhibition was observed, even when the oxidation-reduction potential was maintained at an average Eh of +325 mV by the addition of potassium ferricyanide. When the cultures were aerated, the growth patterns of the three organisms demonstrated different sensitivities to oxygen. P. magnus was found to be the most sensitive. After 2 h of aerobic incubation, no viable organisms could be detected. B. fragilis was intermediately sensitive to oxygen with no viable organisms detected after 5 h of aerobic incubation. C. perfringens was the least sensitive. Under conditions of aerobic incubation, viable organisms survived for 10 h. During the experiments with Clostridium, no spores were observed by spore staining.

Hydrolytic enzymes of anaerobic bacteria isolated from human infections.

Thirty-three strains of anaerobic bacteria isolated from human clinical specimens were examined for the presence of heparinase, hyaluronidase, chondroitin sulfatase, gelatinase, collagenase, fibrinolysin, lecithinase, and lipase activities. Pronounced heparinase activity was limited to species of the genus Bacteroides. A number of species of the genera Bacteroides and Clostridium produced hyaluronidase and chondroitin sulfatase. Gelatinase, collagenase, and fibrinolysin activities were encountered in isolates of the genera Bacteroides, Clostridium, and Peptostreptococcus. All strains capable of degrading collagen also hydrolyzed other protein substrates. Lipolytic activity was minimal among these anaerobic bacteria. No specific hydrolytic activity was consistently associated with the isolates.

Inhibition of Shigella flexneri by the Normal Intestinal Flora III. Interactions with Bacteroides fragilis Strains in Vitro.

Five strains of Bacteroides fragilis isolated from human feces were assayed for their inhibitory activities against Shigella flexneri in vitro. Inhibition was not detected when Shigella and the Bacteroides strains were simultaneously inoculated into a defined liquid medium containing glucose. Inhibition was apparent only when Shigella was inoculated into established Bacteroides cultures. In exponential-phase cultures, the Shigella growth rate was diminished and logarithmic growth was prematurely interrupted. Shigella failed to multiply at all in stationary-phase Bacteroides cultures. An analysis of the stationary-phase cultures revealed that acetic and propionic acids were present in sufficiently high concentrations and the pH of the culture medium was sufficiently low to account for the inhibition of Shigella growth. In glucose-free Bacteroides cultures, Shigella multiplied almost as well as in control cultures, despite the presence of high concentrations of volatile fatty acids. At the high pH levels present in the glucose-free cultures, the acids were not toxic for Shigella. These results reaffirm the importance of the relationship between volatile fatty acids and pH in the inhibition of Shigella growth.

Shigella flexneri inhibition by acetic acid.

The degree of Shigella flexneri inhibition by acetic acid in a chemically defined medium was dependent upon the concentration of undissociated acetic acid in the medium. Under the conditions of the experiments, the critical concentration of undissociated acetic acid that completely inhibited S. flexneri multiplication was approximately 0.0018 M. Adjustment of the medium from pH 6.0 to 7.0 after incubation, which reduced the concentration of undissociated acid 10-fold, completely reversed inhibition, and S. flexneri attained a viable population equivalent to its population in medium without acetic acid. The effects of acetic acid on cellular processes were also studied. The acid interfered with the intracellular accumulation of glucose and glutamic acid but did not interfere with the accumulation of phosphate. The glucose analogue 3-o-methyl-D-glucose, which is taken up by S. flexneri but not metabolized, was used to determine if inhibition resulted from interference with permeation or interference with intracellular accumulation through inhibition of glucose metabolism. Acetic acid did not interfere with the uptake of the glucose analogue by S. flexneri, indicating that inhibition probably involves interference with metabolism. Further evidence for this conclusion was obtained from respiration studies with cell-free extracts in the presence and absence of acetic acid. Inhibition of oxygen uptake by acetic acid in the absence of a permeability barrier suggested a metabolic block rather than interference with permeation. The inhibition of oxygen uptake by cell-free extracts occurred at both pH 6.0 and 7.0, indicating that the degree of dissociation of the acid is unimportant regarding interference with metabolism by intracellular material. The degree of dissociation is important, however, regarding uptake of acetic acid by S. flexneri. Whole cells were more permeable to the undissociated form of acetic acid than to the dissociated form. The data indicate that acetic acid, when taken up by S. flexneri, interferes with the metabolism of glucose by the cells.

Experimental Shigella infections in laboratory animals. I. Antagonism by human normal flora components in gnotobiotic mice.

Germfree mice were associated with selected species of human intestinal bacteria and then challenged with a streptomycin-resistant Shigella flexneri strain. Antagonism against Shigella was most pronounced in mice associated with Escherichia coli and least pronounced in mice associated with Bacteroides fragilis. A moderate degree of antagonism could be demonstrated in mice associated with either Streptococcus faecalis or Bifidobacterium adolescentis. Shigella persisted in the cecal contents of E. coli-associated mice at very low, stable levels. Shigella populations were reduced to levels below detection in the ceca of mice diassociated with E. coli and Bacteroides. Upon subsequent administration of streptomycin, Bacteroides disappeared from the ceca. The E. coli population was greatly reduced, and Shigella reappeared at very high population levels as an apparent recombinant which resembled E. coli biochemically. A streptomycin-resistant E. coli population subsequently emerged and became dominant in the ceca. Shigella concomitantly declined to levels below detection.