Gastrointestinal bacteria generate nitric oxide from nitrate and nitrite

Denitrifying bacteria in soil generate nitric oxide (NO) from nitrite as a part of the nitrogen cycle, but little is known about NO production by commensal bacteria. We used a chemiluminescence assay to explore if human faeces and different representative gut bacteria are able to generate NO. Bacteria were incubated anaerobically in gas-tight bags, with or without nitrate or nitrite in the growth medium. In addition, luminal NO levels were measured in vivo in the intestines in germ-free and conventional rats, and in rats mono-associated with lactobacilli. We show that human faeces can generate NO after nitrate or nitrite supplementation. Lactobacilli and bifidobacteria generated much NO from nitrite, but only a few of the tested strains produced NO from nitrate and at much lower levels. In contrast, Escherichia coli, Bacteroides thetaiotaomicron, and Clostridium difficile did not produce significant amounts of NO either with nitrate or nitrite. NO generation in the gut lumen was also observed in vivo in conventional rats but not in germ-free rats or in rats mono-associated with lactobacilli. We conclude that NO can be generated by the anaerobic gut flora in the presence of nitrate or nitrite. Future studies will reveal its biological significance in regulation of gastrointestinal integrity.     

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 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 (β-glucosidase, β-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.     

Oxidation-reduction potentials in the cecal contents of rats and mice.

The oxidation-reduction potential (ORP) in the cecal contents of conventional rats, germ-free mice, and mice with a Colonization Resistance Factor flora (CRF-mice) was investigated. By using animals that were anaesthetized for a longer period of time, we attempted to eliminate the disturbing influence of oxygen. In addition, measurements were made under anaerobic conditions. For the rats, the ORP values reached a more or less constant level after about 30 min following the insertion of the electrodes. The mean ORP at that time was -458 mV (SD = 45 mV). The mean ORP values for the mice showed a more gradual reduction than was found in the rats. The curves leveled off at about 100 min following the insertion of the electrodes. The mean ORP values 100 min after electrode insertion were: germ-free mice, + 3 mV (SD = 39 mV); CRF-mice, -554 mV (SD = 29 mV). In rats, the ORP value decreased after death; no decrease was observed in mice. No difference was found in the values obtained when measuring under anaerobic or aerobic conditions after death.     

Influence of intestinal microflora on the tryptic activity during lactation in rats

On comparing germ-free and conventional rats, inactivation of the tryptic activity was found to take place in the caecum of conventional adult rats only. A microbial intestinal inactivation of the tryptic activity was established in suckling conventional rats within 10 days after birth. At 3 weeks of age, suckling germ-free rats were found to have less faecal tryptic activity than their early-weaned littermates.     

Degradation of quercetin-3-glucoside in gnotobiotic rats associated with human intestinal bacteria

AIM: The two bacterial species, Eubacterium ramulus and Enterococcus casseliflavus, which had previously been isolated from human faeces using the flavonoid quercetin-3-glucoside as the growth substrate, were tested for their ability to utilize this compound in vivo. METHODS AND RESULTS: Germ-free rats were associated with Eu. ramulus and subsequently with Ent. casseliflavus and vice versa. Identification and enumeration of the bacterial cell counts in faeces and intestinal contents were performed by whole cell fluorescence in situ hybridization. Eubacterium ramulus and Ent. casseliflavus occurred in caecal and colonic contents at cell counts of up to 10(10) g(-1) dry weight. In the jejunum, only Ent. casseliflavus was found (10(9) g(-1) dry weight). Upon oral administration of 32 micromol quercetin-3-glucoside, quercetin was detected in the faeces and urine of germ-free rats (2.2 x 10(-1)-8.1 x 10(-1) micromol 24-h(-1) faeces collection and 1.0 x 10(-2)-2.8 x 10(-1) micromol 24-h(-1) urine collection, respectively) and of rats monoassociated with Ent. casseliflavus (7.9 x 10(-1)-2.7 micromol 24-h(-1) faeces and 1.0 x 10(-1)-5.9 x 10(-1) micromol 24-h(-1) urine, respectively). In contrast, the faeces and urine of rats associated with Eu. ramulus contained 3,4-dihydroxyphenylacetic acid (4.7 x 10(-2)-3.6 micromol 24-h(-1) faeces and 2.4 x 10(-2)-1.0 micromol 24-h(-1) urine, respectively) but only low, or undetectable, concentrations of faecal quercetin (up to 9.3 x 10(-2) micromol 24-h(-1) faeces; detection limit 2.5 x 10(-2) micromol). Urinary quercetin concentrations varied markedly from undetectable amounts up to 1.0 micromol 24-h(-1) urine (detection limit 1.0 x 10(-2) micromol). Isorhamnetin was found in the urine of all animals independent of their bacterial status. There were no significant differences between the groups (2.0 x 10(-2)-2.8 x 10(-1) micromol 24-h(-1) urine). In complete intestinal tissues of animals, associated with both species, quercetin-3-glucoside and its metabolites were detected by a more sensitive and selective method at concentrations that were two to three orders of magnitude lower than in faeces or urine. CONCLUSIONS: These results indicate that Eu. ramulus may be a key organism for the bacterial transformation of flavonoids in the gut.     

Microbial conversion of bilirubin to urobilins in vitro and in vivo

No urobilins are formed from bilirubin in germ-free rats. To isolate and investigate the strains of intestinal microorganisms responsible for this transformation, a suitable test medium was adopted. The strength of the medium and a rather high initial pH were found to be of importance. In this medium, suspensions of rat faeces and a single strain, Cl. ramosum (G62), converted bilirubin to urobilins. Cultivations of Cl. ramosum (G62) together with E. coli significantly enhanced the conversion, whereas addition of 4 other bacterial strains was without the influence. The highest in vitro formation of the urobilins was about 10% of the bilirubin present. When the 6 strains investigated in vitro were established in EXG rats, the in vivo conversion of bilirubin to urobilins was found to be about 15%, compared to 70% in CONV rats.     

乳杆菌喂食大鼠后目标菌克隆筛选与确认

目的确认乳杆菌能否顺利通过胃酸屏障并在肠道内定植为进一步研究乳杆菌对大鼠的生理代谢影响做基础,为乳杆菌菌株的应用提供有效依据。方法采用浓度梯度药物平板筛选利福平耐受菌株,用耐利福平（15 mg/L）乳杆菌菌株喂食SD雄性大鼠并采集其新鲜粪便,在耐药平板上筛选出能在大鼠结肠内定植良好的耐药菌株,并得到16S rDNA的鉴定结果,利用16S rDNA序列同源性分析对乳酸菌进行分类鉴定。结果粪便中检测出的乳杆菌耐药菌株经纯化鉴定后与所喂食的乳杆菌同源。结论实验所筛选出的耐利福平乳杆菌在大鼠结肠内成功定植。     

A note: gut bacteria produce components of a locust cohesion pheromone

AIMS: Faecal pellets from germ-free locusts were used as culture media to determine the ability of locust gut bacteria to synthesize phenolic components of the locust cohesion pheromone. METHODS AND RESULTS: Inoculation of germ-free faecal pellets with Pantoea agglomerans, a species commonly isolated from locusts, resulted in the release of large amounts of guaiacol and small amounts of phenol, both of which are components of the locust cohesion pheromone. Two other locust-derived species, Klebsiella pneumoniae pneumoniae and Enterobacter cloacae, also produced guaiacol from germ-free faecal pellets, but the opportunistic locust pathogen, Serratia marcescens, did not. The most likely precursor for guaiacol is the plant-derived vanillic acid, which is present in large amounts in the faeces of both conventional and germ-free locusts. CONCLUSIONS: These observations are consistent with previous ones, that locust gut bacteria are responsible for the production of components of the locust cohesion pheromone. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings illustrate how an insect can adapt to make use of a common bacterial metabolite produced by one or more of its indigenous gut bacterial species. This observation has implications for our appreciation of insect gut microbiota interactions.     

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.     

Influence of microbial species on small intestinal myoelectric activity and transit in germ-free rats

The effect of an intestinal microflora consisting of selected microbial species on myoelectric activity of small intestine was studied using germ-free rat models, with recording before and after specific intestinal colonization, in the unanesthetized state. Intestinal transit, neuropeptides in blood (RIA), and neuromessengers in the intestinal wall were determined. Clostridium tabificum vp 04 promoted regular spike burst activity, shown by a reduction of the migrating myoelectric complex (MMC) period from 30.5 +/- 3.9 min in the germ-free state to 21.2 +/- 0.14 min (P < 0.01). Lactobacillus acidophilus A10 and Bifidobacterium bifidum B11 reduced the MMC period from 27.9 +/- 4.5 to 21.5 +/- 2.1 min (P < 0.02) and accelerated small intestinal transit (P < 0.05). Micrococcus luteus showed an inhibitory effect, with an MMC period of 35.9 +/- 9.3 min compared with 27.7 +/- 6.3 min in germ-free rats (P < 0.01). Inhibition was indicated also for Escherichia coli X7 gnotobiotic rats. No consistent changes in slow wave frequency were observed. The concentration of neuropeptide Y in blood decreased after introduction of conventional intestinal microflora, suggesting reduced inhibitory control. Intestinal bacteria promote or suppress the initiation and aboral migration of the MMC depending on the species involved. Bacteria with primitive fermenting metabolism (anaerobes) emerge as important promoters of regular spike burst activity in small intestine.     

Determining the viability of faecal bacteria present in germ-free mice

Gram-positive bacilli, originating from the diet, are present in the faeces of germ-free mice in this Unit. Although these organisms have never grown on culture and are assumed to be dead it was considered desirable to test this by non-cultural methods because some gut bacteria are difficult or impossible to grow in vitro by present techniques. Germ-free mice were fed a synthetic diet free from live or dead organisms for 10 days during which time the bacilli disappeared from the faeces, re-appearing when the usual diet was re-introduced. This was regarded as confirmation of the non-viability of the bacilli. The use of Wayson's stain for confirmation of non-viability was found to be inappropriate since it produced false-positive reactions with irradiation-killed bacteria.     

The 'normalization' of germ-free guineapigs with host-specific caecal microflora

Hysterectomy-derived germ-free guineapigs were given colonization-resistant caecal flora from mice (mCRF) or microflora obtained from the caecum of an antibiotic-decontaminated conventional guineapig (gpCRF) and compared with guineapigs raised conventionally with the sow. Body weight and the following intestinal parameters were determined for the groups: colonization resistance (CR) to Escherichia coli, relative caecal weight (RCW), beta-aspartylglycine (faeces), volatile fatty acids (caecum) and bile acids (faeces). mCRF guineapigs showed values quite different from control animals for CR and RCW, indicating the unsuitability of mouse CRF for normalizing guineapigs. In gpCRF guineapigs CR and RCW values were comparable with controls, indicating the suitability of the guineapig flora for normalizing guineapigs. mCRF guineapigs housed with gpCRF guineapigs, showed an improvement in CR and RCW, yielding values found in control animals.     

Characterization of clostridia isolated from faeces of limited flora mice and their effect on caecal size when associated with germ-free mice

115 strains of clostridia were accumulated from 3 separate isolations from the faeces of 1 limited flora (LF) mouse produced by inoculation of germ-free mice with chloroform-treated faeces of conventional mice. These strains were divided into 36 types on the basis of conventional biochemical characteristics. There was some variation in types isolated on the 3 occasions. Although 3 groups of polyassociated (PA) mice were produced from mixing 46 monoassociated mice, each inoculated with 1 of 46 strains of 36 bacterial types, the caecal size of PA mice was still greater than that of the control mice prepared by inoculation of chloroform-treated faeces of an LF mouse. After PA mice of each group were housed together, the caecal size became smaller and was only slightly larger than the control.     

Serum zinc in aging germ-free and conventional rats.

Zinc nutritional status appears to decline with age in humans and rodents. Since germ-free rats outlive their conventional counterparts in better health, serum Zn levels were determined in male germ-free and conventional Lobund Wistar rats in samples originating from the Lobund Aging Study. Starting at 5 months of age, germ-free rats showed significantly higher serum Zn levels than did their conventional counterparts. In conventional rats sacrificed up to 30 months of age in apparently good health, serum Zn levels showed no effect of age, while a slight but significant increase with age was observed in the germ-free rats. In healthy germ-free adults (6-24 months of age), serum Zn concentrations were approximately 25% higher than those in comparable conventional animals. In conventional rats 18-30 months of age (average, 24.5 months), sacrificed because of an obvious moribund condition, serum Zn levels were significantly lower than those in rats of the same age range (average, 24.9 months) that were obviously healthy. Results suggest that the often observed higher absorptive capacity of the germ-free gut might have contributed to higher serum Zn levels, and that a decline in serum Zn concentration with age may be a consequence, rather than a causative factor, of declining health.     

A new technique to determine hydrogen excreted by gnotobiotic rats

A new system, that allowed the monitoring of hydrogen (H2) excretion by gnotobiotic rats without affecting their defined microbial status, was developed. The system consists of an isolator containing a chamber for an experimental animal, and a life-support system (LSS), with a sampling port outside the isolator connected to it. H2 accumulation in the system was measured by analysing a defined volume of gas after removal. H2 concentrations were determined with an electrochemical cell or by gas chromatography. To validate this technique, H2 excretion by germ-free (GF) and mono-associated rats fed a chemically defined diet was measured after oral application of lactulose. Mono-associated rats had been obtained by colonizing GF rats with a H2-producing Clostridium perfringens type A strain isolated from human faeces of a healthy volunteer. Application of 50 mg lactulose to the mono-associated rats resulted in a significant increase in H2 excretion. The net H2 excretion was 7.82+/-1.28 ml H2 in 12 h corresponding to a net maximal rate of 1.1+/-0.3 ml H2/h. In contrast, in experiments with GF rats, less than 0.13 ml H2 were detectable within 12 h. The technique presented is a useful tool for studying bacterial H2 metabolism in vivo under gnotobiotic conditions.     

Variations of brain histamine levels in germ-free and nephrectomized rats

The influence of nephrectomy on brain and peripheral tissue histamine and on brain norepinephrine, dopamine, serotonin, and 5-hydroxyindoleacetic acid was studied in germ-free and conventionally housed rats. The conventional controls had higher levels of histamine in the hypothalamus than the germ-free control animals, but no differences existed for histamine in whole brain minus the hypothalamus or in peripheral tissues. Nephrectomy increased brain histamine and 5-hydroxyindoleacetic acid levels in both germ-free and conventional rats, but had no effect on norepinephrine, dopamine or serotonin. In contrast, the histamine level in the heart of the nephrectomized germ-free animals was lower than that for germ-free controls. There were no changes in the heart or liver histamine levels of the conventional nephrectomized rats.     

Identification of bacteria with beta-galactosidase activity in faeces from lactase non-persistent subjects

Previous studies suggest that, besides the maldigestion of lactose in the small intestine, the colonic processing of lactose might play a role in lactose intolerance. beta-Galactosidase is the bacterial enzyme which catalyzes the first step of lactose fermentation in the colon. We propose a practical method to differentiate and identify bacteria with beta-galactosidase activity in faeces which combines a colony-lift filter assay with X-gal (5-bromo-4-chloro-3-indolyl-beta-d-galactopyranoside) as substrate for differentiation and the fluorescent in situ hybridization technique for identification. The method was applied to faeces from lactase non-persistent subjects. After 28 subjects had undergone one glucose and two lactose challenges, consistent intolerant (n=5) and tolerant (n=7) groups were defined according to their symptom scores. Of the 28 faecal samples, 80.6% (mean, SD: 12.1, range: 47.8-100%) of the total cultured bacteria were found to possess beta-galactosidase activity, which indicates that the bacterial beta-galactosidase is abundant in the colon. The tolerant and intolerant groups did not differ in the percentage or composition of the bacteria with beta-galactosidase activity or beta-galactosidase activity in faeces. Results suggest that the percentage or composition of the bacteria with beta-galactosidase activity in faeces do not play a role in lactose intolerance.     

Morphological effects of high dose neomycin sulphate on the small and large intestine

The morphology of the intestinal wall and the activity of certain mucosal enzyme systems in the course of neomycin treatment were evaluated. Conventional and, to study the role of the bacterial flora, germ-free rats received 500 mg neomycin daily by stomach tube. Rats were sacrificed after seven days and small intestine (proximal and distal part) together with segments of the colon were removed and prepared for histochemistry. The colon and proximal small intestine of untreated conventional and germ-free animals did not show appreciable differences in staining activity after treatment with neomycin. Neomycin diminished both in normal and germ-free rats the activity of NAD tetrazolium reductase, succinate dehydrogenase, esterase, alkaline phosphatase and acid phosphatase in the distal small intestine. The findings of this study indicate that explanations for the beneficial effects of neomycin on hyperammonemia in liver disease should not only include the bactericidal action of neomycin but also its influence on absorption and metabolic functions of the mucosal cells.     

Variation in the incidence of AGUS between different patient populations

OBJECTIVE: To determine the frequency of atypical glandular cells of undetermined significance (AGUS) for three consecutive calendar years from three different referral sources. STUDY DESIGN: Cervicovaginal smears with a diagnosis of AGUS were identified from January 1995 through December 1997. The smears were submitted from three different sources: two were city government hospital clinics, one with predominantly African American and Hispanic patients and the other with predominantly Asian and Hispanic patients. The third referral source was private practitioners' offices with predominantly Caucasian patients. RESULTS: A diagnosis of AGUS was made in 707 cases, accounting for 0.56% of all smears examined. This was in contrast to 6,872 smears reported as atypical squamous cells of undetermined significance (ASCUS) (5.4%) and 3,347 reported as squamous intraepithelial lesions (SIL) or above (2.7%). The incidence of AGUS ranged from 0.16% to 1.00% among different patient populations. This difference was also noted in the rate of ASCUS and SIL in the same patient population. There was a steady increase in the rate of AGUS for each referral source during the study period. The overall rate of patients who underwent histologic evaluation and the incidence of biopsy-proven preinvasive and invasive lesions were 62.4% and 23%, respectively. There was no significant difference in the rate of significant lesions after a diagnosis of AGUS during the study period or between the three referral sources. CONCLUSION: The AGUS rate in our laboratory was low and within the range (0.17-1.83%) reported in the literature. The AGUS rate varies with different patient populations, particularly with the incidence of SIL and age distribution.