A comparative study was conducted on the antibacterial activities of linoleic acid hydroperoxide (LAHPO) and linoleic acid (LA) against the intestinal bacteria including 35 strains, and the representative species were examined for lipid degradation activity. Enterobacterial species (Escherichia coli, Proteus mirabilis, Citrobacter sp. and Enterobacter sp.), Lactobacillus species (acidophilus, casei and plantarum), and Enterococcus faecalis generally showed the same levels in susceptibility to LA and LAHPO. Bacteroides species (distasonis, vulgatus and thetaiotaomicron) and Clostridium ramosum were highly resistant against LA but not LAHPO. Staphylococcus species (aureus and epidermidis), Lactobacillus species (salivarius and fermentum) and the Grampositive anaerobic bacteria including Clostridium perfringens, Bifidobacterium species (bifidum, infantis, adolescentis, breve, longum, animalis, pseudolongum and thermophilus), Peptostreptococcus productus, and Eubacterium sp. were more susceptible to LAHPO than were the enterobacterial species and E. faecalis although they were more resistant against LAHPO than LA. Eubacterium aerofaciens was more susceptible to LAHPO, but strikingly susceptible to both lipids compared to the other bacterial strains. Thin layer chroma-tography showed that C. perfringens degrade LAHPO and C. ramosum degrade LA, which was consistent with the resistance of the corresponding species to the fatty acids. A similar trend in the resistance to and degradation of the fatty acids was found with B. adolescentis and B. infantis. Much higher degradation abilities were observed in the live bacterial cells than the heat-killed cells of B. adolescentis, C. perfringens, and E. coli, suggesting that they can enzymatically degrade LAHPO in vitro.
Experiments were conducted to examine the effect of different vitamin mixture contents in a diet on the cecal microflora and short-chain fatty acid concentrations in male ICR mice. Mice fed a vitamin-free diet showed a profound increase in the numbers of enterobacteriaceae and enterococci and a significant increase in bacteroidaceae in the cecum, while they showed no remarkable changes in the numbers of lactobacilli, bifidobacteria and anaerobic gram-positive cocci. Short-chain fatty acid (SCFA) concentrations were profoundly reduced by the vitamin-free regimen. Mice on a 0.3% vitamin diet showed significant decreases in enterobacteriaceae and enterococci and a significant increase in bifido acteria as compared to animals on a 1% vitamin diet (basal) although their body wcight gain was approximately 60% of the animals fed the basal or 2% vitamin diet. SCFAs showed decreased concentrations; in particular butyrate and valerate were significantly reduced in the 0.3% vitamin group compared to the basal group. The 2% vitamin mixture group showed no significant differences from the 1% vitamin group in cecal microflora composition and SCFA concentrations.
Two encapsulation techniques were applied to Bifidobacterium longum RO23 with the aim of increasing the stability of dried cultures during storage. Commercial freeze-dried cultures of B. longum RO23 were spray-coated with gelatin, xanthan gum or milk fat. The resulting products had water contents which varied from 4.3% (milk fat) to 7.1% water (xanthan gum). Viable counts and acidifying activities of the milk fat-coated cultures were slightly higher than the untreated control after 9 months of storage at 20 °C. The other spray-coated products had lower stability to storage than the untreated control. This might be related to the water contents of the cultures, rather than the coatings themselves, since a correlation was found between water content and storage stability. The addition of oxygen absorbers to 30 ml vials containing 3 g of powder was detrimental to the stability of the cultures. This was potentially due to a 3-4% increase in the water content of the powders under these experimental conditions. Further evaluation of the effect of oxygen absorbers must be made in conditions where water levels remain constant. Cultures of B. longum RO23 were also encapsulated by cocrystallization in lactose. Cocrystallization did not enable a substantial concentration of the culture during formation of the lactose precipitate. Great mortality occurred during the 19 hr crystallization incubation as a 5 log reduction in population was recorded. The survival rate to freeze-drying of the bifidobacteria-containing lactose crystals was estimated at 20%.
The gingival tissues from SUS rats with naturally occurring gingivitis were examined histopathologically. The observations indicated that the inflammation at the early stage was severe neutrophilic infiltration with edema and that the lesion progressed to subacute-chronic inflammation rich in plasma cellular infiltration. Electron microscopic observation indicated that rod-shaped bacteria were present in the connective tissue of gingiva at both inflammation stages. RES rats (control) showed no gingival inflammation and bacterial invasion on the surface of the gingiva. These findings suggested that bacterial invasion can occur at all stages of naturally occurring gingivitis and that it directly affects the gingival tissues.
The inhibitory effects of human-derived Bifidobacterium longum BB536 on harmful intestinal bacteria were examined by co-cultivation of BB536 with each one of the following eight bacterial strains: Escherichia coli, Klebsiella pneumoniae, Clostridium clostridiiforme, C. perfringens, Bacteroides distasonis, B. fragilis, B. thetaiotaomicron and B. vulgatus. In comparison with the results of mono-cultivation, BB536 inhibited both the growth of these putrefactive bacteria and their production of ammonia, and decreased the pH of the culture medium by producing lactic and acetic acids. Enzy-matic assays showed that in BB536 the sorts of the enzymes involved in ammonia production (urease and amino acid deaminases) were rather few and their activities were weaker than observed in the harmful bacteria, whereas the activities of enzymes involved in ammonia assimilation (glutamine synthetase, glutamate synthase and glutamate dehydrogenase) were much higher in BB536 than in the putrefactive bacteria.
Intact cell suspension and/or growing cells of Micrococcus sp. produced a large amount of D-xylonic acid from D-xylose. Utilization of D-xylonic acid by intestinal microorganisms was studied. All 5 species of the genera Bifidobacterium, B. adolescentis M101-4, B. bifidum A234-4, B. breve 153-8, B. infantis I-10-5 and B. longum M101-2, showed good growth on basal medium containing D-xylonic acid. Escherichia coli utilized well Dxylonic acid but not fructooligosaccharides.
Distribution of the fecal microflora in several specimens from different locations of the human stool was analyzed with the steel wool method and the plate-in-bottle method. Total bacterial counts and the number of bacteroidaceae at the upper portion of the stool were significantly higher than those at the lower portion by both culture methods. Lecithinase-negative clostridia were recovered in significantly higher numbers at the middle or upper portion compared with the lower portion by the plate-in-bottle method. The number of lactobacilli was significantly higher at the upper portion than at the other portions. There was no significant difference in the numbers of other bacterial groups observed. The percentage of bifidobacteria in the total bacterial populations enumerated by the steel wool method varied among the three portions of the stool, while those counted by the plate-in-bottle method were consistent. These results indicate that the composition of the fecal microflora was differently analyzed at the parts of the stool possibly because of oxygen diffused from the anus. A culture method for highly oxygen sensitive anaerobes could minimize this difference.
Five non-selective media (medium 10, medium 10 supplemented with 1% extract of eucalyptus leaves, medium 10 with 1% fecal extract of koala, M98-5 and EG agar) by the plate-in-bottle method and two non-selective media (EG agar plate and BL agar plate) by an anaerobic jar method were evaluated for isolation of fecal bacteria in koalas. The predominant isolates were bacteroidaceae, clostridia and eubacteria as obligate anaerobes and enterobacteriaceae as facultative anaerobes. EG agar by the plate-in-bottle method was a suitable medium for isolation of obligate fecal anaerobes from the animals. All the isolates could not be identified to species presently recognized by using currently accepted identification protocols.
We investigated the effects of oral administration of undigestible oligo- and polysaccharides to rats on microbial fermentation products and fecal enzyme activities. The fecal pH values were decreased by feeding diet containing arabinogalactan (AG), kestose (Kes) and nystose (Nys). The fecal moisture was significantly increased by feeding polydextrose (PD), xylooligosaccharide (XO), glucuronosyl xylooligosaccharide (GXO), citrus pectin (CP) and apple pectin (AP) diet. Oligo- and polysaccharides in the diet resulted in a decrease in fecal β-glucosidase, β-glucuronidase and urease activities measured at a constant pH (7.2). PD-containing diet, exceptionally, increased fecal β-glucosidase activity three- to fourfold. Furthermore, fecal enzyme activities were assayed at the fecal pH to assume their actual activities in the colon. These activities were distinctly altered; particularly, β-glucosidase activity in rats fed PD increased and all of the enzyme activities assayed in rats fed Kes and Nys were significantly decreased. These results suggest that the actual enzyme activities of intestinal bacteria should be assayed not at their optimal pHs, but at intestinal pHs. The effects of ten oligo- and polysaccharides on the intestinal environment are discussed in connection with the reduction in the incidence of colon cancer by daily intake of the saccharide.
The viability of freeze-dried cells of bifidobacteria (B. thermophilum ATCC 25525) in broiler starter rations was monitored at different storage temperatures during a twelve-week period. A total of five treatments (control plus four bifidogenic factors), at low (1.55% moisture, aw<0.4) and high water activity (10.52% moisture, aw = 0.64) were included in the study. Results indicated that lower moisture and water activity enhances the survival of bifidobacteria when incorporated into feeds. Refrigeration temperatures (5°C and - 18°C) also increase the survival of bifidobacteria in rations stored for up to twelve weeks. Bifidogenic factors, when included in the feeds, had a limited protective effect on bacterial survival. The use of probiotics, containing bifidobacteria, is an area that needs to be closely evaluated before they can be added to animal rations under commercial conditions.
This paper describes a recommended method for the detection and enumeration of lecithinase-positive clostridia in fecal specimens. The use of specimens treated at 80°C for 10 min in parallel with unheated specimens with both selective and nonselective media is needed for the enumeration of lecithinase-positive clostridia in fecal specimens, when the numbers are less than 104/g.
Lactosucrose (4G-β-D-galactosylsucrose) was fermented in vitro by bifidobacteria and lactobacilli, and to a limited degree by the Bacteroides fragilis group, clostridia, eubacteria, and enterobacteriaceae. The effects of dietary lactosucrose on the fecal flora and fecal metabolites were studied in eight healthy volunteers (20-23 years of age) who ingested 3 g of lactosucrose/day for 7 days followed by 6 g of lactosucrose/day for 7 consecutive days. During lactosucrose intake, the counts of bifidobacteria were increased significantly (p< 0.001), whereas the counts of clostridia, including Clostridium perfringens, and bacteroidaceae were decreased significantly (p<0.05) compared with the values before the intake. The total bacterial counts were decreased significantly (p< 0.05) on day 14 during the intake. The frequency of occurrence of lecithinase-negative clostridia was decreased significantly (p<0.05) when compared with the values before and after the intake. No detectable changes occurred in the counts of other organisms throughout the experimental periods. Fecal concentrations of ammonia, sulfide, phenol, ethylphenol, skatol and indole were decreased significantly (p< 0.05) during lactosucrose intake. Acetic acid and lactic acid were increased significantly (p< 0.05) during the intake. Fecal enzyme activity of β-glucuronidase was decreased significantly (p< 0.05) on day 14 of the intake. Serum very low density lipoprotein (VLDL) was increased significantly (p< 0.01) on day 14 during the intake. Mean fecal pH values decreased from 6.3 to 5.9, and mean water content increased 3.6% during the intake. Fecal weight was increased slightly during the intake. The results obtained showed that the effective dose of lactosucrose for all healthy adults is 3 g/day.
Bifidobacteria are harbored in the intestine of specific animals and humans at different ages. In this study, we examined the immune responses affected by Bifidobacterium longum and B. animalis, focusing upon the cytokines produced by mouse peritoneal cells. Both B. longum and B. animalis induced the expression of IL-1β, IL-6, IL-10 and TNF-α mRNA in mouse peritoneal cells, as determined by RTPCR. The amount of IL-6 secreted by thioglycollate-induced peritoneal exudate cells (TG-PEC) in the presence of these bacteria were similarly augmented, as shown by ELISA. More TNF-α was induced in B. longum than in B. animalis. A difference in the level of TNF production was also suggested from the in vivo tumor suppression test using TNF-sensitive Meth A tumors, although both species of bacteria profoundly suppressed the tumor incidence and tumor growth. Taken together, both B. longum and B. animalis promote the induction and/or secretion of inflammatory cytokines (IL-6 and TNF-α) in mouse peritoneal cells. However, the level of TNF-α production induced by B. animalis was lower than that by B. longum.
Human-flora-associated (HFA) mice produced by oral inoculation with human fecal suspension to germfree mice were examined on the changes of the composition of fecal flora when they were given high-meat (HM) and high-bran (HB) diet and fructooligosaccharides (FOS). The composition of fecal flora of HFA mice was similar to that of the inoculated human feces. Number of enterobacteriaceae significantly increased with HM diet and decreased with HB diet to the level with basal diet. Bacteroidaceae, clostridia and streptococci decreased their numbers with HB diet, while the number of bifidobacteria in HFA mice fed HB diet was significantly higher than those fed HM or basal diet. The rate of bifidobacteria to total bacteria increased and the numbers of bacteroidaceae and enterobacteriaceae tended to decrease in HFA mice administered FOS solution, but the differences were not statistically significant. These results indicate that the changes of floral composition by different dietary components can be observed more obviously in HFA mice than in humans.
We measured the antitumor activity of Bifidobacterium infantis (B. infantis). We used mainly a cell wall preparation (WPG) based upon preliminary experiments (Cancer Res. 45: 1300-1307, 1985). First, we evaluated the antitumor effects of WPG on peritoneal and thoracic tumor-bearing animals, using mouse Ehrlich carcinoma and Meth A fibrosarcoma, as well as rat MADB 106 mammary tumor. More frequent, earlier onset and dose-related injections of WPG were more effective in these tumor-bearing animals. Cell kinetic studies revealed that WPG induced polymorphonuclear cells (PMNs) followed by macrophages at the injection cavity. In addition, WPG directly activated these cells to inhibit the growth of tumor cells in in vitro assays. These results suggest that the bifidobacterial cell wall, WPG induces and activates nonspecific phagocytes (PMNs and macrophages) in situ to reject growing tumor cells in peritoneal and thoracic cavity of animals.
Lactoferrin in feces of healthy individuals was measured by means of latex agglutination method. Lactoferrin titers in 16 out of 31 fecal samples were under 1: 10, and that in 15 were 1: 10 or 1: 20. In the feces with high lactoferrin titer, large numbers of Bifidobacterium were present (more than 1010/g feces); conversely, in the feces with low lactoferrin titer, the numbers of Bifidobacterium were significantly lower (108-109/g feces) (p<0.05). In the feces with high lactoferrin titer, small numbers of Candida were present, but among the samples with low lactoferrin titer, some had large numbers and some had small numbers of Candida. It was demonstrated in vitro that lactoferrin accelerated the growth of Bifidobacterium and slightly suppressed the growth of Candida.
Total 286 strains of lactobacilli were isolated from different segments of the alimentary tract of healthy horses. They were identified by the carbohydrate fermentation patterns and physiological properties as L. salivarius (10 biovars), L. acidophilus (8 biovars), L. reuteri (8 biovars) and Lactobacillus spp.(2 biovars). L. acidophilus and L. reuteri were found in all parts of the alimentary tract, while L. salivarus were not isolated from the stomach. The unidentified Lactobacillus spp. were isolated only from feces.
A total of 19 species of Bifidobacterium were screened for the ability to grow with fructooligosaccharides (FOS) as the carbohydrate source. Several strains of animal origin (B. thermophilum, B. minimum, and B. cuniculi) grew significantly better than strains from human source on inulin (degree of polymerization (DP) ≤ 15). These strains grew equally well on inulin from several sources, but were unable to metabolize levan, a polymer of β (2-6)-D-fructose. Best growth was obtained on FOS preparations containing a significant proportion of low-molecular-weight (DP≥5) carbohydrates. Strains which grew best on inulin required the presence of this FOS for maximal induction of the exo-inulinase. These results suggest that animal strains of bifidobacteria may be used in conjunction with short-chain FOS as a supplement to animal feed.
Hydrolyzate of wheat bran hemicellulose was demonstrated to stimulate significantly the growth of bifidobacteria in the ceca of Wistar rats and ICR mice fed the purified diets including the hydrolyzate at 2.5% and 5.0% for 4 weeks. In contrast, Enterobacteriaceae, viridans streptococci, and staphylococci were decreased in numbers, especially at 5.0% level of the hydrolyzate. Lactobacilli were slightly increased in numbers, but not significantly, in mice. No significant changes were found in the numbers of the other examined microbes. The cecal concentrations of total short-chain fatty acids and acetic and propionic acids increased remarkably in both the animals in proportion to the rate of the hemicellulose hyrolyzate contained in the diets while those of iso-butyric and iso-valeric acids decreased. The butyric acid concentration increased in mice but not in rats. The cecal pH values were inversely proportional to the total concentrations of short-chain fatty acids.
PMC) ; antimicrobial agent-associated diarrhoea (AAD)Clostridium difficile was isolated in 1935 by Hall and O'Toole (29) from the feces of healthy neonate, and the microorganism was first designated Bacillus difficilis.The 'difficilis' was originated from the difficulty in cultivation of the microorganism.They showed that the microorganism had lethality to rabbit or guinea pig and edema-inducing activity.Later the microorganism was classified as Clostridium difficile in 1938 by Prevot (71) .The pathogenicity of C. difficile to human had not been reported until when association of C. difficile with human pseudomembranous colitis (PMC) was demonstrated in 1978.In 1893, Finney (19) first reported an occurrence of PMC in the patient with peptic ulcer after gastroenterostomy.The number of cases with PMC increased in parallel to an increase of use of antibiotics in the 1970s.Although Staphylococcus aureus was speculated to be a causative agent for PMC, there was no determinative evidence for the hypothesis.In 1977, Larson et al (48) reported that clostridial toxin was causative agent for PMC, and in 1978 toxigenic C. difficile was identified as a cause of PMC by Bartlett et al (3), George et al (23) and Larson et al (49) .The isolation rate of C. difficile from feces of healthy adults was 0-12.8%, and that of the patients with PMC was 80-100% (59) .It is considered that change of intestinal flora by administration of antimicrobial agent and following selection of toxin-producing C. difficile are background for the occurrence of PMC.Virulence factors of C. difficile and its pathogenesis in intestinal infection in man are discussed in this review by indicating not only previous reports in the literature but also our own experimental data.VIRULENCE FACTORS OF C. DIFFICILE Virulence factors of C. difficile reported so far are listed in Table 1.Toxins
