Endotoxin in germfree, gnotobiotic, or conventional-flora Sprague-Dawley rats.

The Limulus assay for bacterial endotoxin was performed on serum and (or) plasma from animals monoassociated with Clostridium species, Staphylococcus aureus, Escherichia coli, Proteus mirabilis, Enterobacter agglomerans, Bacteroides fragilis, Klebsiella pneumoniae, or Candida albicans. Plasma from animals monoassociated with the gram-negative bacteria or C. albicans consistently showed a positive Limulus test while conventional-flora controls, germfree rats, and gnotobiotic animals monoassociated with gram-positive bacteria or E. agglomerans were negative. Germfree and conventional rats were injected (intraperitoneal (i.p.)) with Salmonella typhosa lipopolysaccharide (LPS). Although no endotoxin was detectable in either group prior to the injection, by 1 h post injection endotoxin was in the plasma of all groups. The germfree rats appeared to clear the LPS quicker than their conventional-flora counterparts. Generally, LPS-injected rats (conventional and germfree) showed clumping and decreased number of platelets, a decrease in their lymphocyte counts, and increased polymorphonuclear leukocyte (PMN) counts.     

Efficiency of various bacterial suspensions derived from cecal floras of conventional chickens in reducing the population level of Salmonella typhimurium in gnotobiotic mice and chicken intestines

The antagonistic effect exerted towards Salmonella typhimurium by the flora issued from conventional chickens was studied in gnotobiotic animals. In germfree chickens and mice inoculated with S. typhimurium, the highest bacterial counts were observed in ceca, and were not significantly different in either host. The protection afforded by the inoculation of cecal flora issued from a conventional chicken was more effective when this flora was inoculated first into germfree chickens than when it was given only after inoculation with S. typhimurium. Administration of a cecal flora from a 15-day-old chick to gnotobiotic mice and chicken resulted in the inhibition of a further intestinal colonization by S. typhimurium in both hosts. Sixteen strains were isolated among the predominant populations of the fecal flora from chicken flora recipient mice. Association of 14 strains of strictly anaerobic bacteria with 2 strains of Escherichia coli and Streptococcus faecium only decreased the number of S. typhimurium in the ileum of gnotobiotic mice, but not in their cecum. Anaerobe cultures were obtained from 10(-6) and 10(-8) dilutions prepared from the fecal flora of gnotobiotic recipient mice. Antagonistic bacteria were present only in cultures from the 10(-6) dilution. Cecal concentrations of volatile fatty acids were shown not to be the sole factor implicated in the antagonistic effect against S. typhimurium.     

Influence of certain indigenous gastrointestinal microorganisms on duodenal alkaline phosphatase in mice.

Alkaline phosphatase activity was assayed in duodenal homogenates or extracts from adult specific pathogen-free (SPF) and germfree mice and gnotobiotic mice monoassociated with a Lactobacillus sp., a Bacteroides sp., or a coliform strain indigenous to SPF mice. Activity levels of the enzyme were much higher in the preparations from germfree mice than in those from the SPF controls. In the gnotobiotes monoassociated either with a freshly isolated Lactobacillus sp. or a Bacteroides sp., the levels of alkaline phosphatase activity were intermediate between the values for germfree and SPF mice. By contrast, in the gnotobiotes monoassociated with a coliform strain, alkaline phosphatase activity remained at high germfree levels. Butanol extracts of duodenal tissue from SPF mice, germfree mice, and exgermfree mice associated with an indigenous microflora from SPF mice (conventionalized) were subjected to acrylamide gel electrophoresis. A stain for alkaline phosphatase activity revealed three major bands in the gels prepared with extracts from SPF and conventionalized mice, but only two in the gels prepared with extracts from germfree mice. All three bands may have been present in the latter gels. One of the bands (the middle one) may have been obscured, however, by high activity in the slowest moving band. As determined by densitometric scanning, the slowest moving band had much higher activity in the preparations from germfree animals than in those from SPF or conventionalized mice. These findings suggest that the indigenous microbial flora affects not only quantitatively, but also qualitatively, the activity of alkaline phosphatases in the mouse intestinal mucosa.     

Influence of a cecal volume-reducing intestinal microflora on the excretion and entero-hepatic circulation of steroids and bile acids

From mouse fecal material we have isolated four strictly anaerobic bacteria which, when associated with germfree mice or rats, reduced the cecal volume by 80 and 60%, respectively. This cecal volume-reducing flora did not metabolize estrone-3-sulfate, taurolithocholate-3-sulfate or taurolithocholate but gnotobiotic rats associated with this particular flora (CRF-rats) excreted these compounds faster in feces plus urine than did germfree rats. The time needed for 50% excretion (t1/2) of orally administered estrone-3-sulfate was 32 h in germfree rats versus 13 h in CRF rats; for intraperitoneally injected taurolithocholate-3-sulfate the t1/2 was 63 h in germfree versus 17 h in CRF rats and for taurolithocholate the t1/2 was 199 h in germfree and 96 h in CRF rats. Association of germfree rats with the cecal volume-reducing flora did not change the cecal absorption rate of estrone-3-sulfate, but shortened the 50% small intestinal transit time of [14C]PEG from 10 to 3 h; a value also found in conventional rats. These results stress the important influence of the intestinal microflora on the absorption and excretion of steroids via its effect on the physiology of the whole intestinal tract and point to the deficiencies inherent to the use of germfree animals in excretion studies.     

Local Shwartzman phenomenon in germ-free guinea pig]

Eleven germfree and two monoassociated with the Citrobacter guinea pigs, and 25 conventional animals were injected with the heat-inactivated Citrobacter or E. coli for the induction of the local Schwartzman phenomenon. All the gnotobiotic pigs gave a positive reaction. Infiltration at the site of intracutaneous injection was found in all the conventionals, but in none of the gnotobiotics. The data are discussed from the aspect of primary and secondary sequelae of the absence of host microflora.     

Modulation of the rats' immune status by monoassociation with anaerobic bacteria

The capacity of a pure culture of anaerobic intestinal bacteria to influence the host's cellular and humoral immune systems was investigated with germfree, monoassociated, and conventionally reared rats. Monoassociation of germfree rats with Bacteroides fragilis stimulated the production of serum gamma globulin, agglutinating antibodies, and an apparent IgG (immunoelectrophoresis) band. A comparison of the in vitro blastogenic potential of lymphocytes (spleen cells and mesenteric lymph node cells) from germfree, monoassociated, and conventionally reared rats indicated the following: (1) the microbial flora had no obvious effect on the capacity of nonstimulated lymphocytes to incorporate [3H]thymidine; (2) spleen cells from conventionally reared rats responded to phytohemagglutinin, concanavalin A, or pokeweed mitogen better than splenocytes from germfree rats; (3) colonization of germfree rats with Fusobacterium necrophorum increased the responsiveness of splenocytes to photohemagglutinin and concanavalin A; and (4) monoassociation of germfree rats with B. fragilis, but not with F. necrophorum or propionibacterium acnes, increased splenocyte blastogenesis to homologous (i.e., colonizing) bacterial antigens. This study indicated that some intestinal bacteria can modulate the immune status of the host; the extent and nature of this modulation depended on the particular species of colonizing bacteria.     

The effect of sediment redox potential on nitrogen uptake,anaerobic root respiration and growth of Spartina alterniflora loisel

A system developed for growing plants in estuarine sediment at controlled redox potential (Eh) was used to study the effect of Eh on anaerobic root respiration, uptake of added labelled nitrogen and photosynthetic rates of Spartina alterniflora Loisel. Plant growth, estimated by CO₂ fixation and nitrogen uptake, was not affected by sediment redox potential ranging from oxidized (+500 mV) to strongly reducing (?200 mV). Anaerobic root respiration, evident by alcohol dehydrogenase activity, increased with decreasing sediment redox potential. The results suggest that neither redox potential per se nor anaerobic root respiration can account for the growth differences of S. alterniflora observed in Louisiana's coastal marshes.     

Effect of positive redox potentials (>+400mV) on the expression of anaerobic respiratory enzymes in Escherichia coli

The expression of fumarate reductase and other enzymes of anaerobic respiration in Escherichia coli was studied as a function of the redox potential (Eh) in the medium. Redox potentials up to +300 mV allowed full expression of fumarate reductase (frd) genes. Higher values resulted in decreased expression. The relationship between Eh and expression of frd could be approximated by the Nernst equation, assuming a redox couple with a midpoint potential Eo' = +400 mV to 440 mV. At Eh values greater than +510 mV (generated anaerobically by hexacyanoferrate(III] the degree of repression was the same as that obtained by O2. Hexacyanoferrate(III) also caused decreased activities of dimethylsulphoxide (DMSO), nitrite and nitrate reductases. Since expression of these enzymes depends on FNR, the gene activator of anaerobic respiratory genes, it is suggested that the function of FNR is controlled by a redox couple of Eo' = +400 mV to 440 mV.     

抗生素在体内外对厌氧菌和艰难梭菌细胞毒素生成的作用

氯林霉素、灭滴灵和甲砜霉素对大多数肠道厌氧菌的生长具抑制作用。氯林霉素还会破坏肠道菌群平衡,使原来受抑制的艰难梭菌得以定植,并在艰难梭菌浓度达10~8/g盲肠内含物时,检测到艰难梭菌细胞毒素。培养基中亚抑菌浓度的氯林霉素和灭滴灵会推迟艰难梭菌细胞霉素的生成。灭滴灵还可保护无菌小鼠及受氯林霉素处理的悉生小鼠免遭艰难梭菌细胞毒素的致死作用,从而证实了灭滴灵在伪膜性结肠炎临床治疗中的可用性。     

The response of Leuconostoc mesenteroides to low external oxidoreduction potential generated by hydrogen gas

AIMS: The physiological consequences of low external oxidoreduction potential in Leuconostoc mesenteroides were investigated. METHODS AND RESULTS: Leuconostoc mesenteroides was grown under two initial oxidoreduction potential conditions (Eh7: +200 mV and -400 mV) using nitrogen and hydrogen as reducing agents. Growth was affected by Eh7; the lag phase increased from 1 h at an initial Eh7 of +200 mV to 6 h at an initial Eh7 of -400 mV; the maximum specific growth rate at -400 mV was 68% of the one observed at +200 mV. The NADH/NAD+ ratio and (NADH + NAD+) pool were independent of the external Eh7. CONCLUSIONS: This study shows that changing the external oxidoreduction potential from +200 to -400 mV has a strong effect on the Leuc. mesenteroides physiology. The constancy of the maximum carbon and energetic fluxes (qglu, qATP) under the two Eh7 conditions accompanied by the decrease of YX/S and YATP suggested the existence of an uncoupling phenomenon, namely that some catabolized glucose and hence ATP was not associated with biomass production. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper demonstrates the usefulness of taking into account, the effect of the oxidoreduction potential on the growth of Leuc. mesenteroides in the fermentation process.     

Photosynthesis and root growth in Spartina alterniflora in relation to root zone aeration

Spartina alterniflora Lois. is a dominant species growing in intermediate and saline marshes of the US Gulf coast and Atlantic coastal marshes. S. alterniflora plants were subjected to a range of soil redox potential (Eh) conditions representing a well aerated to reduced conditions in a rhizotron system under controlled environmental conditions. The low soil Eh resulted in inhibition of root elongation shortly after treatment initiation. Root elongation was reduced as soil Eh approached values below ca. +350 mV. Substantial decrease in root elongation was noted when soil Eh fell below +200 mV. Generally, net photosynthetic rate (PN) decreased as soil Eh was reduced, with substantial reductions in PN found when Eh approached negative values. Average PN was reduced to 87, 64, and 44% of control under +340, +245, and -180 mV treatments, respectively. The reductions in root elongation and PN in response to low soil Eh indicated the adverse effects of low soil Eh on plant functioning and the need for periods of soil aeration that allow plants to resume normal functioning. Thus periods of drainage allowing soil aeration during the growing season appear to be critical to S. alterniflora by providing favorable conditions for root growth and gas exchange with important implications for plant carbon fixation.     

Transit time of epithelial cells in the small intestines of germfree mice and ex-germfree mice associated with indigenous microorganisms.

Germfree mice housed in isolators under controlled environmental and nutritional conditions were associated with an intestinal microflora. These associated animals and germfree mice drawn from the same population were tested for the rate at which the epithelial cells transited from the crypts of Lieberkuhn to the tips of the villi in their small intestines. The method for estimating the rate of transit of epithelial cells involved the use of liquid scintillation counting to determine the amount of radioactivity entering the cells while the animals were being injected with [3H]thymidine and statistical analysis of th data with a computer program developed for the purpose. As estimated by that method, the cells transited from the crypts to the villous tips in germfree mice in about 115 h and in the associated animals in about 53 h. In monoassociated mice, a strain of a Lactobacillus sp. had no effect on the transit time of the epithelial cells. A strain of Torulopsis pintolopesii stimulated uptake of 3[H]thymidine by the small bowel mucosae in mice monoassociated with the organisms for 5 weeks. In animals monoassociated with the yeast fo 3, 4, and 6 weeks, however, the radioactive compound was incorporated into the bowel mucosae to the same extent as the mucosae of germfree mice. Therefore, similarly to the Lactobacillus strain, T. pintolopesii has no obvious influence on the transit rate of small bowel epithelial cells.     

Absence of cecal secondary bile acids in gnotobiotic mice associated with two human intestinal bacteria with the ability to dehydroxylate bile acids in vitro.

Germ-free mice were orally inoculated with human intestinal 7alpha-dehydroxylating bacterial strains to evaluate their ability to transform bile acids in vivo. Three weeks after inoculation of the bacteria, cecal bile acids were examined. Among free-form bile acids, only beta-muricholic acid was detected in the cecal contents of gnotobiotic mice associated with Bacteroides distasonis strain K-5. No secondary bile acid was observed in the cecal contents of any of the gnotobiotic mice associated with 7alpha-dehydroxylating bacteria, Clostridium species strain TO-931 or Eubacterium species strain 36S.     

Elevated levels of colloid osmotic pressure in cecal contents of germfree animals.

Determinations of colloid osmotic pressure in the supernatant of germfree rat cecal contents indicated substantially elevated values in comparison to those of rat blood plasma or of conventional rat cecal supernatant. The germfree cecal supernatant, under conditions of similar total osmolality, was able to draw water at a sizable rate from a polyvinylpyrollidone solution whose colloid osmotic pressure was taken to be equivalent to that of interstitial fluid. It is suggested that the water absorption inhibition which was observed in the lower bowel of germfree rodents, is in part caused by the colloid osmotic pressure gradient which exists in these animals between the luminal contents and the tissue component.     

Effect of Bacterial Contamination on Cecal Size and Cecal Contents of Gnotobiotic Rodents

In the present investigation the effect of various bacterial contaminations of gnotobiotic mice and rats on cecal size is presented. Of the species tested, Bacteroides oralis and Fusobacterium nucleatum did not establish in germ-free mice. Streptococcus mutans, Clostridium difficile, a Neisseria strain and two recent cecal isolates established, but failed to exert an effect upon the cecum of mice. A group K streptococcus and B. fragilis increased the cecal size apparently by increasing the levels of water-soluble protein, peptides, and carbohydrates in the cecal contents. Mixed ileal bacteria decreased the cecal size by preventing accumulation of soluble proteins and carbohydrates in the cecum. A Peptococcus strain caused a reduction by lowering the levels of insoluble material in the cecum. When this strain was combined with two Clostridium isolates and introduced into gnotobiotic rats, 50 to 65% cecal reduction was observed. This polycontamination did not decrease the per cent water of the cecal contents but caused lower levels of both soluble and insoluble material to accumulate in the cecum. No net nitrogen absorption from the distal small intestine occurred in either the germ-free or polycontaminated rats.     

The effect of sediment chemistry on the successful establishment of wild rice (Zizania palustris L.) in northern Saskatchewan water bodies

Sediment chemistry of productive and non-productive wild rice sites was measured over 3 consecutive growing seasons Although productive sites differed from non-productive sites in terms of pH, N and potential N mineralization, redox potential was considered to be the major factor which distinguished these site classes. Productivity of wild rice in commercial stands could not be consistently correlated to sediment nutrients. However, growth appeared to be restricted in strongly reduced sediments and sediment Eh is considered to be a rapid, accurate method of estimating site potential. Good growth is expected if Eh is above –150 mB while poor growth is expected when Eh drops below –200 mV. Eh values of sediment samples changed as storage time increased. It is therefore recommended that Eh is recorded on-site.     

The effect of sediment redox potential and soil acidity on nitrogen uptake,anaerobic root respiration,and growth of rice (Oryza sativa)

Summary Oryza sativa Loisel cultivar Mars., a common lowland rice variety was grown under controlled soil redox conditions (Eh) and acidity (pH). The effect of two variables (Eh and pH) on growth, anaerobic root respiration, and uptake of added labelled nitrogen, was investigated. Plant growth, estimated by dry weight showed significantly higher growth under reducing sediment redox potentials (−200 mV and 0 mV) and at a soil pH of 6.5 Using the activity of the inducible enzyme alcohol dehydrogenase (ADH) as an indicator of anaerobic root respiration, a decrease in redox potential resulted in an increase in root ADH. However, growth paralled increases in anaerobic root respiration suggesting nitrogen transformation in the soil to be a primary parameter governing growth. Labelled nitrogen uptake which was greater under anaerobic conditions apparently led to greater growth of lowland rice in the highly reduced or anaerobic soil treatments.     

Photosynthesis and root growth in Spartina alterniflora in relation to root zone aeration

Spartina alterniflora Lois. is a dominant species growing in intermediate and saline marshes of the US Gulf coast and Atlantic coastal marshes. S. alterniflora plants were subjected to a range of soil redox potential (Eh) conditions representing a well aerated to reduced conditions in a rhizotron system under controlled environmental conditions. The low soil Eh resulted in inhibition of root elongation shortly after treatment initiation. Root elongation was reduced as soil Eh approached values below ca. +350 mV. Substantial decrease in root elongation was noted when soil Eh fell below +200 mV. Generally, net photosynthetic rate (PN) decreased as soil Eh was reduced, with substantial reductions in PN found when Eh approached negative values. Average PN was reduced to 87, 64, and 44% of control under +340, +245, and -180 mV treatments, respectively. The reductions in root elongation and PN in response to low soil Eh indicated the adverse effects of low soil Eh on plant functioning and the need for periods of soil aeration that allow plants to resume normal functioning. Thus periods of drainage allowing soil aeration during the growing season appear to be critical to S. alterniflora by providing favorable conditions for root growth and gas exchange with important implications for plant carbon fixation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Distribution and effects of a defined six-member murine-derived microflora in gnotobiotic gerbils.

The gnotobiotic gerbil was selected as a model with which to study the effects of colonization with a defined microflora on organ morphology, histology, and selected blood biochemical parameters. Gerbils were maintained germfree for 13 months but failed to reproduce, presumably because of the enlarged cecum. A colony of gnotobiotic gerbils that was associated with a bacterial flora consisting of Lactobacillus brevis, Streptococcus faecalis, Staphylococcus epidermidis, Bacteroides vulgatus, Enterobacter aerogenes, and a Fusobacterium sp. was established. These gnotobiotic gerbils had smaller ceca than germfree gerbils and proved capable of reproduction. Except for the presence of large numbers of Bacteroides organisms in the stomach and greater numbers of S. epidermidis in gnotobiotic gerbils, the number and location of gastrointestinal bacteria were similar in conventional and gnotobiotic gerbils. Bacteroides sp. was the second most predominant microorganism present in gnotobiotic gerbils, whereas clostridia were reported to be the second most predominant microorganism in conventional gerbils. Microscopic examination of direct-impression smears indicated that fusobacteria were present on mucosal surfaces. Intestines of gnotobiotic gerbils weighed twice as much as the intestines of conventional gerbils. Intestinal tissue water weight values from conventional and gnotobiotic gerbils were similar. Histological examination of gerbil intestinal tissue revealed no cellular hypertrophy and no evidence of inflammation in gnotobiotic gerbil intestines. Spleens of gnotobiotic gerbils showed no germinal center stimulation. Statistical differences in total serum glucose, serum protein, and hematocrit levels were found between conventional and gnotobiotic gerbils.     

Colonization of gnotobiotic mice by Roseburia cecicola, a motile, obligately anaerobic bacterium from murine ceca.

In scrapings of mouse cecal mucosae, motile bacteria outnumbered nonmotile bacteria by a ratio of 2:1. Obligately anaerobic bacteria were obtained from such scrapings through the use of techniques designed for the selective isolation of motile bacteria. One of the isolates, Roseburia cecicola, was rapidly motile in broth by means of 20 to 35 flagella arranged in a fascicle on each cell. R. cecicola cells colonized germfree mice (3 x 10(9) to 1 x 10(10) CFU/g of cecum) within 11 days after the animals were inoculated intragastrically with 2 x 10(8) CFU per mouse. In such monoassociated gnotobiotes, the bacteria were found primarily in the cecum, dispersed in the lumen among particles of digesta, and in the mucus over the epithelial surface. Between 2 and 3 weeks after birth, offspring of monoassociated adult mice were colonized by the bacterium (2 x 10(9) to 1 x 10(10) CFU/g of cecum). These results indicate that R. cecicola is suitable for studies of the ecology of host-associated microorganisms, particularly for investigation of the role of motility and possibly also chemotaxis in bacterial colonization of the mammalian gastrointestinal tract.