Establishment of a biochemically active intestinal ecosystem in ex-germfree rats

A time course study for the establishment of some biochemical microbial intestinal functions was undertaken in ex-germfree rats conventionalized, i.e., colonized with conventional flora, in three different ways: untreated (group 1); contact with visitor rats (group 2); inoculated with intestinal contents from conventional rats (group 3). The first two groups of rats were inoculated with the intestinal contents from conventional rats after being out of the germfree isolators for 4 weeks. The biochemical parameters studied were degradation of mucin, inactivation of tryptic activity, conversion of cholesterol to coprostanol and of bilirubin to urobilinogen, degradation of beta-aspartylglycine, and formation of short-chain fatty acids. The results showed that the way in which the microbes were introduced and the microbial biochemical functions themselves were of importance. In several cases, social contacts, i.e., contact with visitor rats, were just as effective for the functionally adequate establishment of microbial intestinal functions as was inoculation with intestinal contents from conventional rats. Some of the biochemical parameters studied were established after a few days, whereas the establishment of others was markedly delayed. When inoculated after 4 weeks, all rats in the first two groups were colonized with conventional flora within 1 week. The results indicate that the model system described is suitable when studying buildup mechanisms in intestinal ecosystem(s).     

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.     

重构肠道菌群Ⅱ型糖尿病大鼠造模血脂代谢变化及肝细胞线粒体超微结构观察

目的重构肠道菌群Ⅱ型糖尿病大鼠模型脂类代谢变化比较,探讨肠道菌群潜在的调脂作用。方法将64只SD雄性大鼠随机分为对照组、造模组、益生组和去污组。采用高糖高脂喂养,亚致病剂量（30mg／mL）链脲佐菌素腹腔一次性注射造模,分别于2周、4周后取腹腔主动脉血做血脂检测,同时处死大鼠取肝脏做电镜切片观察。结果注射4周后各组出现的胆固醇升高两两比较差异有统计学意义（P〈0．01）,LDL升高差异有统计学意义（P〈0．01）;益生组肝细胞线粒体受损明显;去污组血脂升高相对较轻,肝细胞内有大量脂肪堆积。结论益生菌对Ⅱ型糖尿病大鼠胆固醇升高有一定改善作用,肝线粒体损伤较明显;去污染组损伤相对较轻,而脂肪堆积明显;提示糖尿病脂类代谢与肠道菌群密切相关。     

Trypsin and chymotrypsin activity of the intestinal content in germfree, monoassociated and conventional rabbits.

Trypsin (T) and chymotrypsin (CHT) activities in luminal contents of the ileum, caecum and sigmoideum were followed in conventional (6 animals), monoassociated (5) and germfree (5) rabbits by pH-stat automatic titration using p-toluenesulphonyl-L-arginine methylester and acetyl-L-tyrosine ethylester as substrates. In conventional rabbits with complete microbial flora an aborally increasing decline of both proteolytic activities of luminal contents was determined (ileum T 198.2 - CHT 100.0; signmoideum T 10m.2 - CHT 68.8 mrg/g of intestinal content). Monoassociated animals represent a group different from both germfree and conventional animals. Trypsin and chymotrypsin of intestinal contents were not significantly altered by the presence of megacaecum in germfree rabbits (ileum T 219.2 - CHT 160.2; sigmoideum T 208.8 - CHT 110.8 mug/g of intestinal content). Chymotrypsin in the intestinal contents appears more labile and more affected by microbial flora than trypsin.     

Intestinal colonization of laboratory rats with Oxalobacter formigenes.

Six strains of Oxalobacter formigenes (anaerobic oxalate-degrading bacteria) were examined for their ability to colonize the gastrointestinal tracts of adult laboratory rats. These rats did not harbor O. formigenes. Strain OxCR6, isolated from the cecal contents of a laboratory rat that was naturally colonized by oxalate-degrading bacteria, colonized the ceca and colons of adult rats fed a diet that contained 4.5% sodium oxalate. Five days after rats were inoculated intragastrically with 10(9) viable cells of strain OxCR6, oxalate degradation rates in cecal and colonic contents increased by 19 and 40 times, respectively. Viable counts of strain OxCR6 from these rats averaged 10(8)/g (dry weight) of cecal contents. Strain OxCR6 was not detected in the cecal contents of inoculated rats fed diets that contained less than 3.0% sodium oxalate. Strains of O. formigenes isolated from the cecal contents of swine, guinea pigs, and wild rats and from human feces also colonized the ceca of laboratory rats; a ruminal strain failed to colonize the rat cecum.     

Expression of mitochondrial HMGCoA synthase and glutaminase in the colonic mucosa is modulated by bacterial species.

The expression of the colonic mitochondrial 3-hydroxy 3-methyl glutaryl CoA (mHMGCoA) synthase, a key control site of ketogenesis from butyrate, is lower in germ-free (GF) than in conventional (CV) rats. In contrast, the activity of glutaminase is higher. The objective of this study was to investigate whether the intestinal flora can affect gene expression through short chain fatty acid (SCFA) and butyrate production. GF rats were inoculated with a conventional flora (Ino-CV) or with a bacterial strain producing butyrate (Clostridium paraputrificum, Ino-Cp) or not (Bifidobacterium breve, Ino-Bb). In the Ino-CV rats, mHMGCoA synthase expression was restored to the CV values 2 days after the inoculation, i.e. concomitantly with SCFA production. In the Ino-Cp group, but not in the Ino-Bb group, mHMGCoA synthase and glutaminase were expressed at the level observed in the CV rats. These data suggest that the intestinal flora, through butyrate production, could control the expression of colonic mHMGCoA synthase and glutaminase. These modifications in gene expression by butyrate in vivo seem unrelated to a modification of histone acetylation.     

DNA-methyltransferase activities in Streptomyces antibioticus

Abstract To quantitate ammonia production by the intestinal flora, ammonia levels in arterial blood and the venous effluent of the small and large bowel of conventional, selectively decontaminated, germ-free and gnotobiotic rats were measured.
When the anaerobic flora was removed by decontamination a significant decrease in ammonia levels was observed in the effluent of both the small and large intestine. Decontamination of aerobic flora did not result in depression of ammonia production. Gnotobiotic rats colonised with an anaerobic flora or with a mixed aerobic and anaerobic flora, showed a slight increase in ammonia levels. No increase in ammonia production was observed when rats were colonised with aerobic flora. These results indicate that the Enterobacteriaceae were not responsible for ammonia generation.
The increase in ammonia levels after colonisation with anaerobic or mixed anaerobic/aerobic flora did not completely restore ammonia levels, despite reaching bacterial counts which were comparable to those in conventional rats. This may be explained by the limited number of species with which the rats were colonized. The finding that aerobic flora does not significantly contribute to ammonia production suggests that neomycin, known to be exclusively effective against aerobic flora, must have other effects to explain improvement of hepatic encephalopathy.     

Effect of Methanobrevibacter sp MF1 inoculation on glycoside hydrolase and polysaccharide depolymerase activities, wheat straw degradation and volatile fatty acid concentrations in the rumen of gnotobiotically-reared lambs

Four naturally born lambs were placed in sterile isolators 24 h after birth before the natural establishment of cellulolytic microorganisms and archaea methanogens. At the age of 6 weeks they were inoculated with pure cultures of the strains FD1 and 007 of Ruminococcus flavefaciens and at the age of 4 months with a pure culture of Methanobrevibacter sp. MF1. Following the establishment of MF1, the population of R. flavefaciens slightly increased in the rumen of the four lambs, there was also an increase in straw degradation, in the activity of some glycoside and polysaccharide hydrolases of the adherent microbial populations and in the concentration of acetate in ruminal contents.     

Studies on Streptococci

The population levels of bacteria in the contents and the walls of the gastrointestinal tract of gnotobiotic rats inoculated with lactic acid bacteria (streptococci, lactobacilli, and bifidobacteria) from humans and rats were determined. Lactobacilli as well as streptococci isolated from rats colonized in the digestive tracts of the gnotobiotic rats at a high population level, characteristically highest in the stomach. On the other hand, in the rats inoculated with human lactic acid bacteria, streptococci were dominant in the lower tract. The human lactobacilli or bifidobacteria did not colonize when the organisms in each genus were inoculated together with streptococci. However, when all three genera were inoculated together, lactobacilli and bifidobacteria colonized. Observations on the species of streptococci showed that the intestinal type of streptococci was found to colonize at a high population level, but the oral type was not. Strains of the same genus of lactic acid bacteria from humans and from rats showed different colonization patterns.     

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).     

Changes in colonic mucins of germfree rats in response to the introduction of a "normal" rat microbial flora. Rat colonic mucin.

In order to determine the influence of bacterial colonization on amount and composition of colonic mucins, germfree male AS/Ztm rats were colonized with a rat specific intestinal flora for different times (2, 7, 14, 21, 28, 35, 120 days). The amount of colonic mucins was determined by gel filtration on Sepharose CL-4B; the relative amount of acidic mucins was calculated after ion exchange chromatography. In addition, cecal weight and dry matter of feces were monitored. While germfree and SPF rats revealed similar amounts of colonic mucins (7.0 vs. 7.2 mg mucin/300 g body weight), the initial phase of association was characterized by considerably decreasing values. After four weeks of association, the total amount of colonic mucins had almost equalized in the two groups. The amount of acidic mucins, having decreased during the first three weeks of colonization, rendered values comparable to the SPF mucins after four months of adaptation. Cecomegaly in germfree rats disappeared within the first two days, while solidification of the intestinal content occurred within four months. Mucin losses during initial phase of association are attributed 1. to the disappearance of the cecal mucin pool, and 2. to the mucin degrading activity of some bacterial strains known to be present in the intestinal flora. Further development is conducted by a stimulation of mucin secretion, described to follow the colonization. The initially increased secretion of neutral mucins is attributed to a pronounced release of immature mucin glycoproteins, while the shift to more acidic mucins is considered to result from stimulated secretion as well as from a selective bacterial degradation of neutral mucin components.     

Gastrointestinal pH measurement in rats: influence of the microbial flora, diet and fasting

The pH of the rat intestinal tract was decreased by the presence of a microbial flora, but its influence in the forestomach is less clear. Stomach pH values varied according to the amount of food present at the time of measurement. Fasting increased the pH of the gastrointestinal tract in conventional rats but had little effect in germfree rats. In the conventional rat, feeding a purified diet compared with a commercial diet resulted in a lower pH in the forestomach and a higher pH in the caecal contents. Magnesium trisilicate promoted gastric emptying in conventional rats and its antacid effect was observed only in the caecum and colon.     

Colonization resistance against Pseudomonas aeruginosa in gnotobiotic mice

Gnotobiotic (GB) mice were colonized with various groups of intestinal bacteria to determine which members of the indigenous flora would exert colonization resistance against Pseudomonas aeruginosa. P. aeruginosa was cultured from the faeces at levels of 10(3)-10(4) cells/g in GB mice inoculated with either the combination of bacteroides and clostridia obtained from conventional (CV) mice or the combination of bacteroides, lactobacilli and clostridia obtained from limited flora mice. The combination of lactobacilli and clostridia from CV mice also did not eliminate P. aeruginosa from GB mice. However, P. aeruginosa was not detected in the faeces of GB mice by 14 days after inoculation with the combination of bacteroides, lactobacilli and clostridia obtained from CV mice. Thus, a complex indigenous flora consisting of bacteroides, lactobacilli and certain clostridia obtained from CV mice but not clostridia obtained from limited flora mice is required to exert complete colonization resistance against P. aeruginosa in GB mice.     

痛泻要方对肝气乘脾泄泻小鼠肠道微生物活度的影响

目的探讨痛泻要方对肝气乘脾泄泻小鼠肠道微生物活度的影响,为痛泻要方通过调节肠道微生物治疗肝气乘脾泄泻提供实验基础和理论依据。方法将25只SPF级雄性昆明小鼠随机分为正常组10只和模型组15只,造模成功后,随机选取正常组和模型组动物各5只,在无菌环境下提取肠道内容物和肠黏膜,剩余模型动物随机分为自然恢复组和治疗组各5只,治疗成功后处死各组动物提取肠道内容物和肠黏膜。用荧光素二乙酸法测定各样品中微生物活度。结果肝气乘脾泄泻造模小鼠肠道内容物微生物活度高于正常组,与正常组相比差异有统计学意义(P=0.01),而模型组小鼠肠黏膜微生物活度降低,与正常组相比差异有统计学意义(P=0.01)。痛泻要方治疗组和自然恢复组小鼠肠道内容物微生物活度均降低,与正常组相比差异无统计学意义(P>0.05),治疗组和自然恢复组小鼠的肠黏膜前段和后段均与正常组差异有统计学意义(P=0.001),肠黏膜中段自然恢复组与正常组差异有统计学意义(P=0.043),而治疗组与正常组没有差异。结论肝气乘脾泄泻造模使肠道内容物微生物活度增加,而使肠黏膜微生物活度降低。痛泻要方能够使肝气乘脾泄泻小鼠肠道内容物中微生物活度降低,恢复至正常水平,增加肠黏膜中段微生物活度,对肠黏膜后段微生物活度增加作用更显著。     

新生儿肠道菌群的动态观察及临床意义

本文以厌氧菌双歧杆菌和需氧菌大肠杆菌为代表对29例健康足月新生儿(阴道分娩儿13例、剖腹产分娩儿16倒)生后1周内肠道细菌的定植,进行了动态观察。结果表明:两组新生儿首先肠道定植细菌是大肠杆菌,后为双歧杆菌,而且阴道分娩儿肠道优势菌双歧杆菌定植及达优势化时间均早于剖腹产分娩儿。提示,分娩方式也是影响肠道细菌定植的因素之一,为临床治疗和护理提供了参考。     

Establishment of specific pathogen-free guinea-pig colonies using limited-flora guinea-pigs associated with conventional guinea-pig flora, and monitoring of their cecal flora.

Six groups of limited flora (LF) Hartley guinea-pigs were produced by inoculation of hysterectomy-derived GF guinea-pigs with various combinations of cecal bacteria of conventional (CV) guinea-pigs to determine the effective bacterial cocktails for the establishment of a specific pathogen free (SPF) colony. Bifidobacterium magnum (Bif) isolated from CV guinea-pigs was used for pretreatment. The mortality of LF guinea-pigs inoculated with only Bif was 75%, and that of those inoculated with Bif plus chloroform-treated cecal suspension (CHF) or Bif plus CHF plus 32 isolates from CV guinea-pigs was 40 to 66.7%. These three groups were in an unhealthy condition with mucoid enteritis-like diarrhea. However, the mortality of LF guinea-pigs inoculated with the anaerobic growth on EG plates injected with 10(-5) dilution of cecal contents (CF) or inoculated with Bif plus CF was 6.3 and 15%, respectively. These latter two groups of LF guinea-pigs were transferred to separate barrier rooms and some of the LF guinea-pigs were maintained in isolators as a source of intestinal flora for SPF guinea-pigs. The composition of cecal flora of LF guinea-pigs was stable for a long time, and bacteroidaceae and peptococcaceae were maintained as predominant components. The basic composition of the cecal flora of SPF guinea-pigs originated from LF guinea-pigs, which consists mainly of the anaerobic bacteria, was not changed over a long period, and the flora composition became similar to that in CV guinea-pigs. Guinea-pig-specific pathogens from the SPF colonies were not detected during experiments.     

Effect ofMethanobrevibactersp MF1 Inoculation on Glycoside Hydrolase and Polysaccharide Depolymerase Activities, Wheat Straw Degradation and Volatile Fatty Acid Concentrations in the Rumen of Gnotobiotically-reared Lambs

Four naturally born lambs were placed in sterile isolators 24 h after birth before the natural establishment of cellulolytic microorganisms and archaea methanogens. At the age of 6 weeks they were inoculated with pure cultures of the strains FD1 and 007 ofRuminococcus flavefaciensand at the age of 4 months with a pure culture ofMethanobrevibactersp. MF1. Following the establishment of MF1, the population ofR. flavefaciensslightly increased in the rumen of the four lambs, there was also an increase in straw degradation, in the activity of some glycoside and polysaccharide hydrolases of the adherent microbial populations and in the concentration of acetate in ruminal contents.     

Influence of conventionalization on small-intestinal mucosa of germ-free Wistar rats: quantitative light microscopic observations

Germ-free rats were inoculated with microbial flora from feces of conventionally reared rats and the mucosal structure was quantitatively observed at different time intervals after the inoculation and at different regions of the small intestine. In the ileum, desquamation figures were frequently seen on the villus tip, and several parameters of the mucosal elements, i.e., villus and crypt lengths, mitotic figures, goblet cells and thickness of lamina propria were significantly increased after the inoculation. On the other hand, in the duodenum and jejunum, such parameters except for the lamina propria showed no remarkable change during the course of the experiment, though the villus/crypt ratio increased temporarily at half a day after the inoculation. These regional differences of the mucosal response to the inoculation may be due to the different populations of microbial flora which settled in each region of the small intestine.     

Microbial colonization of human ileal conduits

Morphological and microbiological techniques were used to locate and identify the microorganisms that colonized the human ileal conduits in 17 different patients from 5 days after surgery up to as many as 16 years of service as a urine conduit. The ecological sequence of this colonization assumes some practical importance because the ascending growth of pathogenic organisms in this essentially open, unvalved urinary tract diversion system leads to the development of life-threatening pyelonephritis. Extensive examination of the microvillus surfaces of the ilea of five accident victims by both transmission and scanning electron microscopy showed that these tissue surfaces were not colonized by bacteria, even in the absence of prophylactic antibiotic therapy, and that these surfaces were not occupied by adherent microorganisms after several years of service as a urine conduit, even when the skin surface stoma and the conduit contents were heavily colonized by bacteria and yeasts. During the initial period (10 days) of postoperative antibiotic therapy, the mucus and urine within the conduit were largely colonized by yeasts. A mixed population of yeasts and gram-positive cocci subsequently developed in the conduit itself, and gram-positive cocci were seen to be avidly adherent to epidermal cells at the stoma. As antibiotic protection was gradually withdrawn, gram-negative organisms became a part of the mixed microbial flora of the conduit contents, and some of the potentially pathogenic organisms of this group (e.g., Escherichia spp., Proteus spp., Pseudomonas spp., etc.) were isolated from patients with pyelonephritis that appeared to come from the ileal conduit.(ABSTRACT TRUNCATED AT 250 WORDS)