Glycosphingolipid patterns of the gastrointestinal tract and feces of germ-free and conventional rats

Acid and non-acid glycosphingolipids of stomach, small and large intestine, and stimulated feces of germ-free and conventional rats of the same stain have been isolated and characterized. The glycosphingolipid patterns of the intestinal organs were chemically and immunologically very similar between the two groups of rats and relatively unaffected by the presence of an intestinal microbial flora. The major exception was the presence of hematoside with N-glycoloylneuraminic acid (NeuGc) (NeuGc alpha 2----3Gal beta 1----4Glc beta 1----1Cer) in the stomach of conventional rats not found in the stomach of germ-free animals. Glycosphingolipids of stimulated feces of germ-free animals were derived from epithelial cells mainly of the small intestine and showed no signs of degradation. Glycosphingolipids of feces of conventional rats completely retained the pattern of blood group A-, B-, and H-active glycolipids as found in sterile feces but contained less of hematoside and more of lactosylceramide. This effect was probably due to degradation by bacteria, as demonstrated in vitro with the production of lactosylceramide after treatment of the isolated acid glycolipids of sterile feces with neuraminidase from Clostridium perfringens. The amount of total non-acid glycosphingolipids per dry weight was similar for stomach, was 50% higher for small intestine, and 300% higher for large intestine of germ-free animals compared to conventional animals. Due to the presence of large amounts of mucins the dry sterile feces contained 12% less non-acid glycolipids than conventional feces. However, calculated per rat per day the germ-free animal excreted more of non-acid glycosphingolipids (1.8 and 1.2 mg, respectively).     

阿莫西林干预对婴儿优势菌在无菌小鼠体内定植的影响

目的考察阿莫西林干预对婴儿优势菌双歧杆菌及乳杆菌在无菌小鼠体内定植的影响。方法 1日龄Balb/c无菌乳鼠接种婴儿粪便悬液。饲养至7~21日龄灌胃阿莫西林(100mg/kg),对照组在同日龄给予等体积的生理盐水。利用qRT-PCR检测小鼠粪便中双歧杆菌、乳杆菌的含量。结果阿莫西林处理可显著降低乳杆菌(P0.05)、双歧杆菌(P0.05)在无菌小鼠体内定植数量,但停药后饲养至成年(53日龄)二者定植数量与对照组小鼠比较差异无统计学意义(P0.05)。结论哺乳期阿莫西林干预会导致乳杆菌、双歧杆菌在小鼠体内定植量下降,但停药后小鼠饲养至成年二者可达到正常定植量,婴儿菌群定植小鼠模型可以模拟与现有动物模型一致的阿莫西林对双歧杆菌、乳杆菌定植的影响规律。     

Mice and Monkeys as Assay Animals for Clostridium perfringens Food Poisoning

Spores and vegetative cells of Clostridium perfringens, in combination with meat or starch paste, sterile culture filtrates, lecithinase, and phosphorylcholine, were administered to mice and rhesus monkeys in an attempt both to evaluate the animals as test agents and, if possible, to elucidate the active factors producing food-poisoning symptoms caused by this organsim. Some of the preparations were administered to the monkeys by stomach tube; others, in gelatin capsules which were treated with formaldehyde so that the release of their contents was delayed and presumably reached the intestines of the animals. Any changes in intestinal passage times and in consistency of stools of the animals were observed, and the counts of C. perfringens in the feces of the monkeys previous and subsequent to treatment were recorded. The results obtained were inconclusive. Diarrhea occurred only relatively infrequently in both species, regardless of the substance fed or the mode of administration. The changes in intestinal passage times were not great, although in the monkeys there appeared to be a slight trend toward reduction as the magnitude of the bacterial load increased. Phosphorylcholine appeared to have little, if any, effect in reducing intestinal passage time of mice or monkeys. No procedures explored in these experiments could be said to be satisfactory as a means of animal assay for food poisoning strains of C. perfringens since typical symptoms did not appear with regularity.     

阿莫西林干预IHFA小鼠及对其成年后肠道菌群的影响

目的利用婴儿菌群人源化小鼠(IHFA小鼠)观察阿莫西林对其干预后及成年后肠道菌群的影响。方法新生Balb/c无菌小鼠接种纯母乳喂养的婴儿粪便获得IHFA小鼠。7~21日龄灌胃给予100mg/kg阿莫西林,对照组给予等量的生理盐水。采用变性梯度凝胶电泳(DGGE)检测小鼠在21日龄及53日龄的肠道菌群。结果 21日龄的阿莫西林处理组IHFA小鼠肠道菌群与正常对照组比较差异有统计学意义(P<0.05);即使在停药后饲养至53日龄的成年小鼠,阿莫西林处理组小鼠肠道菌群仍然存在细微差异。结论哺乳期治疗剂量阿莫西林处理不仅严重干扰小鼠肠道菌群结构,同时导致其成年后肠道菌群不能完全恢复。     

Effects of Age and Strain on the Microbiota Colonization in an Infant Human Flora-Associated Mouse Model

The establishment of human flora-associated animal models allows the in vivo manipulation of host, microbial, and environmental parameters to influence the gut microbial community. However, it is difficult to simulate infant gut microbiota in germ-free animals because of the variation and dynamic state of infant microbial communities. In this study, the effects of age and strain on intestinal microbiota were observed in an infant human flora-associated (IHFA) mouse model. To establish an IHFA model, postnatal day (PND) 1 germ-free mice (Kunming, n = 10; BALB/c, n = 10) were infected with feces from a breast-fed infant. Microbiota in the feces of BALB/c mice (at PND 7, 14, and 21), and Kunming mice (at PND 14) were analyzed by PCR-denaturing gradient gel electrophoresis. Bifidobacteria and lactobacilli levels in the feces of BALB/c and Kunming mice (PND 7/14/21) were detected by quantitative real-time PCR. The Dice similarity coefficient (Cs) for the fecal microbiota of IHFA mice in comparison with the HD donor sample was higher for BALB/c mice than for Kunming mice (P < 0.05). In addition, the DCs at PND 7 were lower than those at PND 14 and PND 21 in both mouse strains (P < 0.05). The Bifidobacteria and Lactobacillus species colonizing the BALB/c mice were similar to those in the Kunming mice (at PND 7/14/21). The bifidobacteria counts increased with age in both mouse strains, whereas the lactobacilli counts decreased with age in both strains. These results suggest that both age and strain influence microbiota patterns in the IHFA mouse model.     

Saccharomyces boulardii stimulates sIgA production and the phagocytic system of gnotobiotic mice

The effect of Saccharomyces boulardii on the immune system was evaluated, comparing germ-free Swiss/NIH mice monoassociated with the probiotic with germ-free mice. Saccharomyces boulardii colonized the gut of germ-free mice and survived the gastrointestinal conditions. An increase in sIgA production, both total and anti-S. boulardii, was observed in the intestinal contents of monoassociated mice when compared with germ-free controls. The number of Kupffer cells was significantly higher in monoassociated mice than in germ-free controls. In S. boulardii-monoassociated mice, clearance of Escherichia coli B41 was higher than in germ-free controls. TNF-alpha, IFN-gamma and IL-12 serum levels were higher at earlier time points in monoassociated mice when compared with germ-free mice. These results show that the yeast S. boulardii modulates the host immune responses. This effect may be of interest for improving the resistance to enteropathogenic bacterial infections.     

Growth and Invasiveness of Candida albicans in the Germ-Free and Conventional Mouse After Oral Challenge

Candida albicans was established in large numbers throughout the gut after one oral challenge in the germ-free and in the conventional mouse. Of the strains tested, only the germ-free ND 1 mouse appeared to be susceptible to infection, and this was confined to the stomach mucosa; lesions contained large numbers of hyphal and mycelial forms with blastospores. These forms were also seen in the gut of resistant germ-free ND 4 mice after challenge. Only budding yeast forms were seen in the gut contents from conventional animals. The concentration of sulfhydryl-containing compounds was decreased in the stomach contents from germ-free mice. The stomach tissue of conventional animals seemed to be more acidic than that of germ-free animals, and association of C. albicans with conventional mice neutralized some of this acidity. E(h) values of contents from the gut of unchallenged mice were usually higher in conventional than in germ-free animals; after challenge, the E(h) in both groups decreased. Some reciprocal effects of intestinal microorganisms and host are discussed in relation to intestinal candidiasis.     

Response of White Mice to Cells and Culture Constituents of Clostridium perfringens

Broth cultures of Clostridium perfringens (ATCC 10543) were fractionated by ammonium sulfate precipitation and Sephadex G-150 chromatography. Components isolated, as well as some enzymes present in the culture, were assayed for toxicity by feeding to white mice. Early work indicated that when a meat-fat-starch slurry, infected with C. perfringens, was fed to mice, the intestinal passage time was reduced. By using large numbers of mice as test animals and analyzing the data statistically, we found that C. perfringens and several fractions from the culture supernatant significantly affected the mice. A toxic material present in the supernatant was not identifiable as phospholipase C. Phospholipase C and physphorylcholine affected the intestinal passage time of the mice only when large amounts were given. The enzyme, neuraminidase, and another unidentified compound present in the supernatant affected the passage time when very small amounts were fed to mice.     

Antisense-RNA-mediated decreased synthesis of small, acid-soluble spore proteins leads to decreased resistance of clostridium perfringens spores to moist heat and UV radiation

Previous work has suggested that a group of alpha/beta-type small, acid-soluble spore proteins (SASP) is involved in the resistance of Clostridium perfringens spores to moist heat. However, this suggestion is based on the analysis of C. perfringens spores lacking only one of the three genes encoding alpha/beta-type SASP in this organism. We have now used antisense RNA to decrease levels of alpha/beta-type SASP in C. perfringens spores by approximately 90%. These spores had significantly reduced resistance to both moist heat and UV radiation but not to dry heat. These results clearly demonstrate the important role of alpha/beta-type SASP in the resistance of C. perfringens spores.     

Biochemical differentiation between enterotoxigenic heat-sensitive and heat-resistant Clostridium perfringens strains

Some biochemical characteristics of 37 enterotoxigenic Clostridium perfringens strains isolated from human feces, ground beef, and soil samples by heat-selection methods and of two NCTC strains were studied. Two different biochemical patterns closely related to the heat resistance of the strains were found. The strains placed into group 1 were trehalose, inositol, and sorbitol negative and synthesized heat-resistant spores, while those placed into group 2 were trehalose and inositol positive and synthesized heat-sensitive spores. Sorbitol fermentation was variable among the strains of this last group. The strains of group 1 were more cellobiose, melibiose, and salicin fermentative than those of group 2. Only the strains placed into group 2 synthesized toxins of sufficient levels for typing. In spite of having been isolated by mild heat treatment of the specimens, two strains showed the same biochemical and toxigenic characteristics of the strains of group 1. The heating of these two strains did not modify their characteristics. We conclude that enterotoxigenic C. perfringens strains showing the two different toxigenic and biochemical patterns are present in the human gut, ground beef, and, probably, in soil. These strains may be differentiated on the basis of their capacity to produce acid from trehalose, inositol, and sorbitol, heat resistance of the spores and grade of toxigenicity. The heat-selection methods used for isolation of C. perfringens strains from different sources exerted a selection of strains from one or another group, but had no influence on their toxigenic and biochemical properties.     

Antagonistic effect exerted by three strictly anaerobic strains against various strains of Clostridium perfringens in gnotobiotic rodent intestines

Our purpose was to study bacterial antagonism between a limited number of strictly anaerobic strains and Clostridium perfringens in the intestinal tract of gnotobiotic rodents. Gnotobiotic mice harboring a Bacteroides thetaiotaomicron, a Fusobacterium necrogenes, and a Clostridium sp. strain were protected against pathogenic B, C, and D C. perfringens serotypes. A drastic antagonistic effect of this three-strain association was also observed against a nonpathogenic C. perfringens serotype A (CpA). It was less efficient in gnotobiotic rats than in mice and less efficient in gnotobiotic mice fed an autoclaved diet than in mice fed the same diet sterilized by irradiation. No diffusible inhibitory substances against CpA were detected in feces of gnotobiotic mice harboring the three antagonistic strains, and no nutrient depletion was demonstrated in filtrates prepared from 10-fold diluted feces of these mice. In vitro mixed cultures of the three antagonistic strains failed to inhibit growth of CpA, whereas CpA did not multiply in a 10-fold diluted feces from gnotobiotic mice. A reverse correlation between the initial number of antagonistic strains and the division number of CpA was determined using serially diluted fecal suspensions. Thus, large numbers of viable cells of both antagonistic strains were required to inhibit the target strain in fecal suspensions as was also found in gnotobiotic mice intestines. However, no diffusible inhibitory substance was detectable nor could depletion of growth factors be identified as causing antagonism. Whatever factors that may be responsible for antagonism were found to be influenced by the host and its diet.     

Identification and Typing of Clostridia in Raw Milk in Egypt

S ummary : Three spp. of clostridia were isolated from samples of raw cow and buffalo milk obtained near Cairo. Of 150 isolates of anaerobes, 108 were Clostridium perfringens , 30 were Cl. butyricum , and 12 were Cl. sporogenes. The Cl. perfringens isolates comprised 100 nonhaemolytic and 8 pathogenic haemolytic strains. The latter strains typed by neutralization tests, were of type A. Fifteen of the nonhaemolytic strains were also of type A; of these, 6 strains produced heat resistant spores and 9 strains produced heat susceptible spores. Feeding mice with these 15 nonhaemolytic strains caused marked reduction in intestinal passage time.     

beta-Aspartylglycine, a substance unique to caecal contents of germ-free and antibiotic-treated mice.

The caecal supernatants from germ-free, antibiotic-treated and control mice were compared with respect to their content of low-molecular-weight substances (less than 3500 mol. wt.). The supernatants contained about the same amount of free amino acids. After acid hydrolysis, the caecal supernatants of germ-free and antibiotic-treated mice showed a 2.9-fold increase in free amino acids, whereas a similar treatment of the supernatant from control mice resulted in a 2.6-fold increase. By gel filtration on Sephadex G-25, and high-voltage paper electrophoresis at pH 3.5 of the fractions eluted after the void volume, it was found that the caecal supernatants of germ-free and antibiotic-treated mice contained a substance more acidic than aspartic acid. Preparative high-voltage electrophoresis, dansylation, amino acid analysis and a specific colour reaction showed the substance to be beta-aspartylglycine. After a minimal 36 h of treatment with neomycin and bacitracin, a high concentration of beta-aspartylglycine was found, and no enterococci and aerobic Gram-negative rods could be cultured from the caecal contents. The possibility that in one mouse the appearance of beta-aspartylglycine was related to a decrease in Gram-negative rods was ruled out by selective elimination of aerobic Gram-negative rods by using polymyxin B. This suggests that other bacteria concomitantly eliminated with the enterococci and aerobic Gram-negative rods, directly or indirectly, could play a role in the accumulation of beta-aspartylglycine.     

The suitability of Tórtora's medium for the production of enterotoxin in Clostridium perfringens strains

Examination of 200 samples from soil and the same number of samples from healthy human feces yielded 49 (24.5%) and 105 (52.5%) strains of heat-resistant Clostridium perfringens respectively. Fourteen (7.0%) strains isolated from soil and 37 (18.5%) from feces synthesized enterotoxin, as demonstrated by Tórtora's method, at sufficient levels to permit its detection by mouse lethality, microslide double gel diffusion or counterimmunoelectrophoresis tests. By using the Duncan-Strong (DS) method, only four (2%) enterotoxigenic strains from soil and 14 (7.0%) from feces were obtained. The supernatant fluid from two enterotoxigenic-negative strains grown in DS medium gave a false-positive reaction when they were injected intravenously into mice. Tórtora's medium was preferable because a larger number of isolated strains produced spores and enterotoxin to permit their recognition as enterotoxigenic strains.     

The use of a rat-derived microflora for providing colonization resistance in SPF rats

To obtain a suitable species-specific microflora for a new rat SPF-unit, germ-free WAG/Rij rats were associated with a flora derived originally from selectively decontaminated Cpb: WU (Wistar) rats. Caecal and ileal contents of these rats had been cultured anaerobically (37 degrees C) for 7 days and harvested. This cultured flora was given to germ-free Cpb: SE (Swiss) mice, which were kept in an isolator system and acted as a source of the flora to associate germ-free Wag/Rij rats. In these associated rats, several parameters indicative of the 'quality' of the intestinal microflora were investigated and compared to those in rats with a mouse derived anaerobic microflora. Parameters included relative caecal weight, colonization resistance and the concentration of faecal bile acids. The cultured rat-derived microflora normalized the observed intestinal parameters better than the mouse derived microflora, and provided better colonization resistance. We conclude that culturing of intestinal contents of selectively decontaminated animals can be a useful way to obtain a species-specific donor-microflora which can be used to start new SPF units.     

Reversal of radiation-dependent heat sensitization of Clostridium perfringens spores.

The effect of solute concentration on the sensitization of Clostridium perfringens spores to heat by ionizing radiation was investigated. As we have shown previously, spores of C. perfringens treated with gamma radiation are now sensitive to subsequent heat treatments than are spores that receive no radiation treatment. When gamma-irradiated spores were heated in the presence of increasing concentrations of glycerol or sucrose, the heat sensitivity induced by irradiation was progressively decreased. The magnitude of the increase in heat resistance induced by extracellular solutes was greater in gamma-irradiated spores than in nonirradiated spores. Based on these observations, it is proposed that the induction of heat sensitivity in spores by radiation is related to the loss of osmoregulatory or dehydrating mechanisms in irradiated spores.     

Reversal of radiation-dependent heat sensitization of Clostridium perfringens spores

The effect of solute concentration on the sensitization of Clostridium perfringens spores to heat by ionizing radiation was investigated. As we have shown previously, spores of C. perfringens treated with gamma radiation are now sensitive to subsequent heat treatments than are spores that receive no radiation treatment. When gamma-irradiated spores were heated in the presence of increasing concentrations of glycerol or sucrose, the heat sensitivity induced by irradiation was progressively decreased. The magnitude of the increase in heat resistance induced by extracellular solutes was greater in gamma-irradiated spores than in nonirradiated spores. Based on these observations, it is proposed that the induction of heat sensitivity in spores by radiation is related to the loss of osmoregulatory or dehydrating mechanisms in irradiated spores.     

Intestinal sphingolipid excretion associated with feeding of phytohemagglutinin lectin (Phaseolus vulgaris) to germ-free and conventional rats

Intestinal sphingolipids of feces of germ-free and conventional rats were analyzed during the pair feeding of a complete defined diet containing phytohemagglutinin lectin (PHA) from red kidney beans (Phaseolus vulgaris) as 1% dietary protein in comparison to casein fed controls. Phytohemagglutinin in the diet increased the total fecal excretion of sphingomyelins (18-fold for germ-free and 20-fold for conventional rats), of non-acid glycosphingolipids (3.5-fold for germ-free and 9-fold for conventional rats) and also of the gangliosides (2.5-fold) for the germ-free rats compared to controls. For germ-free rats the increase of non-acid glycolipids was ascribed to an effect of the lectin strictly on the small intestinal mucosa, while for conventional rats an effect was seen also on the large intestinal mucosa. Increase of fecal gangliosides of germ-free rats was due mainly to an increased excretion ofN-acetylneuraminosyl-lactosylceramide, a ganglioside species restricted to epithelial cells of duodenum, of upper jejunum and of large intestines. The effects on glycolipid excretion observed in germ-free rats and the rather similar effects seen in conventional animals suggested that the influence of dietary PHA was due directly to effects elicited by PHA binding to the enterocyte brush border membrane and not to secondary effects induced by increase in the luminal microflora.     

Comparison of the levels of heat resistance of wild-type, cpe knockout, and cpe plasmid-cured Clostridium perfringens type A strains

An enterotoxin (cpe) plasmid was cured from a Clostridium perfringens non-food-borne gastrointestinal disease (NFBGID) isolate, and the heat resistance levels of wild-type, cpe knockout, and cpe plasmid-cured strains were compared. Our results demonstrated that (i) wild-type cpe has no influence in mediating high-level heat resistance in C. perfringens and (ii) the cpe plasmid does not confer heat sensitivity on NFBGID isolates.     

Detection of viruslike particles in germ-free mice

Kajima, Masahiro (University of Notre Dame, Notre Dame, Ind.), and Morris Pollard. Detection of viruslike particles in germ-free mice. J. Bacteriol. 90:1448-1454. 1965.-Viruslike particles similar in structure to the mouse leukemia agent were detected by electron microscopy in thymus tissues of germ-free AK mice which had developed spontaneous leukemia. In addition, unique viruslike particles (type B) were detected in tissues from spontaneous mammary adenocarcinoma of germ-free C3H mice. Leukemia virus-like particles were also observed in the thymuses of the control AK mice and of the C3H control mice as well as of those with mammary tumors. Germ-free mice are not virus-free. The routes of transmission of leukemia as well as of mammary tumor viruses may be "vertical," through the embryo or placenta.