The effect of enterococci on normalizing the intestinal microflora in experimental dysbacteriosis

The normal microflora of the intestine produces essential influence on the vital activity of the host. The exposure of the body to the action of different unfavorable factors (roentgen radiation, the administration of antibiotics, Salmonella infection, etc) results in changes in the normal microflora of the gastrointestinal tract. This work was aimed at the study of the influence of Streptococcus faecium YDC-48 on the intestinal microflora of mice in experimental (chemotherapeutic, postirradiation) dysbacteriosis and Salmonella infection. The effect of the oral administration of S. faecium YDC-48 on the correction of the intestinal microflora of mice in cases of dysbacteriosis etiology was studied. The intragastric administration of S. faecium YDC-48 was found to induce an increase in the level of lactobacterin and a decrease in the number of opportunistic microorganisms in chemotherapeutic and postirradiation dysbacteriosis. The oral administration of S. faecium YDC-48 decreased the manifestations of intestinal dysbacteriosis in experimental Salmonella infection. The possibility of developing a preparation on the basis of S. faecium YDC-48, a representative of normal intestinal microflora, is discussed.     

Influence of antibiotics on some intestinal microflora associated characteristics

Intestinal functions related to the presence of microbes in host organisms are normally heavily influenced by administration of antimicrobial drugs. We have investigated the effect of several antibiotics in man and rat, on some MACs (Microflora Associated Characteristics). A MAC is defined as the recording of any anatomical structure, biochemical or physiological function in the host organism which is influenced by microflora. When functional, active microbes are absent as in germfree animals, healthy newborns, or in relation to antimicrobial therapies, a MAC defined as a GAC (Germfree Animal Characteristic). Faecal samples have been collected prior to, during and up to several weeks after the antimicrobial administration in order to investigate different MAC/GAC patterns. Microbial conversion of cholesterol to coprostanol, bilirubin to urobilinogen and 7-alpha-dehydroxylation of cholic acid have been investigated to evaluate hepatic/intestinal co-functions, and degradation of intestinal mucin in order to evaluate the integrity in the intestinal mucosa. Furthermore, degradation of the dietary derived beta-aspartylglycine, the level of faecal trypsin and production of short chain fatty acids were investigated to evaluate dietary/intestinal co-functions. It is concluded that each antimicrobial drug creates its own profile, both with regard to duration and function.     

Influence of oral doxycycline therapy on the diversity and antibiotic susceptibility of human intestinal bifidobacterial population

Aim:  To evaluate the influence of doxycycline therapy on the composition and antibiotic susceptibility of intestinal bifidobacteria.
Methods and Results:  Faecal samples were collected from nine subjects receiving doxycycline therapy and ten control subjects, and analysed for bifidobacteria by culturing and PCR–DGGE (denaturing gradient gel electrophoresis). A marked decrease in the diversity (average number of amplicons detected by PCR–DGGE 0·8 in the antibiotic vs 4·3 in the control group) of Bifidobacterium populations was observed during doxycycline therapy. The proportion of a tetracycline-resistant bifidobacterial population was higher in the antibiotic group than in the control group (83% vs 26%). Based on the tet gene PCR, resistance could be associated with the presence of tet (W). In two subjects, strains representing highly similar genetic fingerprints but different tetracycline susceptibilities were detected. A mutation causing lack of functionality in the tet (W) was observed in one of the susceptible strains.
Conclusions:  Doxycycline therapy had a drastic effect on the diversity and tetracycline susceptibility of intestinal Bifidobacterium populations.
Significance and Impact of the Study:  The use of broad-spectrum antibiotics increased the pool of tetracycline-resistant commensal bacteria in the intestine. The detection of resistance genes alone is not sufficient for the evaluation of bacterial antibiotic resistance.     

肠道微生物菌群变化对接受肠内营养支持方案的 颅脑损伤患者手术愈后的影响

目的研究肠道菌群对重度颅脑损伤术后肠内营养患者的影响。方法选择重度颅脑损伤患者40例,全部给予肠内营养制剂。根据病人的愈后和生存情况,分为恶化组和康复组。诊断病人的感染情况并采用qPCR检测肠道菌群情况。结果颅脑损伤病人术后,恶化组GCS评分要明显高于康复组。恶化组肠有害菌(大肠杆菌和肺炎克雷伯菌)的相对表达水平显著高于康复组,而有益菌(粪肠球菌、乳酸菌和双歧杆菌)的表达水平显著低于康复组。结论肠道微生物分布对接受肠内营养的重度颅脑损伤手术后患者有影响,有益菌的存在对其愈后有促进作用。     

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.     

幽门螺杆菌感染和抗菌治疗对食道下端菌群的影响

目的通过小鼠动物模型,探讨幽门螺杆菌（Helicobacter pylori,H.pylori）感染和用抗生素抗H.pylori治疗对食道下端菌群的影响,分析其与食道下端疾病发生的关系。方法将45只Balb/c小鼠随机均分为阴性对照组,感染组和治疗组。感染组和治疗组通过灌喂H.pylori建立动物感染模型,治疗组再灌喂奥美拉唑,氨苄青霉素,克拉霉素根除H.pylori。三组小鼠均在用抗生素处理后同时处死,取食道下端组织提取细菌的DNA,以原核生物16S rDNA V6区通用引物采用聚合酶链发应-变性梯度凝胶技术（PCR-DGGE）检测,用Quantity One1-DAnalysis software对DGGE图谱进行菌群结构分析,并将DGGE图谱上的组间差异条带用16S rDNA V6区引物分别扩增后,DNA测序,BLAST比对鉴定。结果成功制备了小鼠幽门螺杆菌感染模型,用抗生素有效根除了H.pylori感染。小鼠食道下端DGGE指纹图谱分析显示,各组间条带数量比较差异均有统计学意义（P〈0.05）,多样性指数、丰富度指数差异有统计学意义（0.05〉P〉0.001）,均匀度指数差异无统计学意义（P〉0.05）。菌群聚类分析能很好地在相似性进化树中分开,主成分分析不同组的菌群分别聚集在不同的位置,BLAST比对分析对照组,感染组具有特有细菌。结论食道下端定植着由大量细菌构成的较稳定的菌群,H.pylori感染与治疗后菌群结构有明显变化。     

Influence of intestinal microflora on the amino acid composition of lamb feces]

6 conventional and 5 germfree male lambs were fed ad libitum a UHT sterilized cow milk. Body weight and food intake were recorded. Whole feces were collected for 5 consecutive days. Growth rate reached 259 g/d for the germfree. Daily fecal excretion of dry matter and nitrogenous compounds are not found different in the two groups of animals. The influence of intestinal microflora appears on the biochemical composition of the feces. As compared to the conventional fecal proteins from germfree lambs are very high in threonine and serine and low in lysine. Moreover the difference of amino acid composition between these two groups come not only from the histidine alanine and arginine composition of bacteries; it also involves the high levels of threonine serine cystine and tyrosine of the endogenous digestive proteins.     

西藏灵菇奶对抗生素相关性腹泻小鼠肠道菌群的调整作用

目的研究用西藏灵菇对抗生素引起的肠道菌群失调及病理变化的调整作用.方法由林可霉素造成小鼠腹泻模型,并证实肠道菌群紊乱后,将其分成灵菇奶治疗组和自然恢复组,7 d后对每个组小鼠进行肠道菌群检测,同时取回肠末段组织标本用光镜及电镜观察黏膜结构的变化.结果抗生素模型组大多数小鼠肠道黏膜出现不同程度的损害,小鼠肠道的正常菌群及病理组织改变基本恢复正常.结论灵菇奶不仅对肠道菌群有调整作用,而且对肠黏膜的修复有一定作用.     

正常人肠道肠球菌耐药性与生物膜形成关系的初探

目的了解正常人肠道肠球菌对临床常用抗生素的耐药水平和其生物膜的形成情况,并初步探讨肠球菌的耐药性与其生物膜形成之间的关系。方法用K-B法测定正常人肠道肠球菌对15种抗生素的敏感性,用96孔聚苯乙烯板进行生物膜形成试验。结果生物膜形成阳性菌株对高浓度链霉素、四环素和红霉素的耐药性(耐药率分别为42.9%、90.5%、71.4%)显著高于生物膜形成阴性的菌株(耐药率分别为4.8%、38.1%、42.8%),对其余12种抗生素的耐药性与生物膜形成阴性株差异无统计学意义。结论生物膜形成对肠球菌耐药性增强有一定作用,但还与其本身耐药性和抗生素的性质有关。     

Starch utilization by the human large intestinal microflora

High levels (2–565 units/g) of amylase activity were observed in human faeces. Over 92% of amylase activity in faeces obtained from healthy persons was extracellular, whereas only about 9% of activity was associated with particulate material and washed cells. Bacterial cell-bound amylases were considerably more efficient in breaking down starch, however, than were the soluble enzymes which occurred in cell-free faecal supernatant fluids. Cell population densities of anaerobic starch-hydrolysing bacteria in the stools of ten persons ranged from 1.1 × 10¹⁰ to 3.3 × 10¹²/g of faeces. Identification of 120 starch-hydrolysing colonies isolated from the stools of six subjects showed that the predominant amylolytic bacteria belonged to the genera Bifidobacterium, Bacteroides, Fusobacterium and Butyrivibrio. Mixed populations of gut bacteria rapidly fermented starch with the production of volatile fatty acids and organic acids. Lactate was observed to be a major, though transient intermediate during starch fermentation by these cultures. Approximately 60% of starch utilized was converted to volatile fatty acids, which in the human colon would be potentially available for absorption.     

Starch utilization by the human large intestinal microflora

High levels (2-565 units/g) of amylase activity were observed in human faeces. Over 92% of amylase activity in faeces obtained from healthy persons was extracellular, whereas only about 9% of activity was associated with particulate material and washed cells. Bacterial cell-bound amylases were considerably more efficient in breaking down starch, however, than were the soluble enzymes which occurred in cell-free faecal supernatant fluids. Cell population densities of anaerobic starch-hydrolysing bacteria in the stools of ten persons ranged from 1.1 X 10(10) to 3.3 X 10(12)/g of faeces. Identification of 120 starch-hydrolysing colonies isolated from the stools of six subjects showed that the predominant amylolytic bacteria belonged to the genera Bifidobacterium, Bacteroides, Fusobacterium and Butyrivibrio. Mixed populations of gut bacteria rapidly fermented starch with the production of volatile fatty acids and organic acids. Lactate was observed to be a major, though transient intermediate during starch fermentation by these cultures. Approximately 60% of starch utilized was converted to volatile fatty acids, which in the human colon would be potentially available for absorption.     

Metabolism of 6-nitrochrysene by intestinal microflora

Since bacterial nitroreduction may play a critical role in the activation of nitropolycyclic aromatic hydrocarbons, we have used batch and semicontinuous culture systems to determine the ability of intestinal microflora to metabolize the carcinogen 6-nitrochrysene (6-NC). 6-NC was metabolized by the intestinal microflora present in the semicontinuous culture system to 6-aminochrysene (6-AC), N-formyl-6-aminochrysene (6-FAC), and 6-nitrosochrysene (6-NOC). These metabolites were isolated and identified by high-performance liquid chromatography, mass spectrometry, and UV-visible spectrophotometry and compared with authentic compounds. Almost all of the 6-NC was metabolized after 10 days. Nitroreduction of 6-NC to 6-AC was rapid; the 6-AC concentration reached a maximum at 48 h. The ratio of the formation of 6-AC to 6-FAC to 6-NOC at 48 h was 93.4:6.3:0.3. Interestingly, compared with results in the semicontinuous culture system, the only metabolite detected in the batch studies was 6-AC. The rate of nitroreduction differed among human, rat, and mouse intestinal microflora, with human intestinal microflora metabolizing 6-NC to the greatest extent. Since 6-AC has been shown to be carcinogenic in mice and since nitroso derivatives of other nitropolycyclic aromatic hydrocarbons are biologically active, our results suggest that the intestinal microflora has the enzymatic capacity to generate genotoxic compounds and may play an important role in the carcinogenicity of 6-NC.     

益生菌及人体肠道菌群对低聚甘露糖的降解

目的研究益生菌及肠道菌群对低聚甘露糖的降解情况。方法(1)将5名志愿者粪便样品,13株纯菌以及7种微生态制剂接种到VI-MO液体培养基中,37℃厌氧培养24或72h,取样并采用TLC(薄层层析)检测降解情况;(2)选取7种微生态制剂中降解能力较强的乐塞益生菌胶囊(LS)中的益生菌作为筛选源,采用VI-MO固体培养基分离纯化低聚甘露糖的单一降解菌;(3)将B.uniformis L8和L.plantarum LS1A同时接种到VI-MO液体培养基中,37℃厌氧培养72h,取样并采用TLC检测降解情况。结果 (1)不同人体肠道菌群和微生态制剂对低聚甘露糖的利用情况存在较大差异,B.uniformis L8和B.xylanisolvens C5可利用大聚合度低聚甘露糖;(2)从微生态制剂中分离得到的L.plantarum LS1A可利用小聚合度低聚甘露糖;(3)B.uniformis L8利用MO产生的小寡糖能被L.plantarum LS1A利用。结论低聚甘露糖被拟杆菌降解释放出的小聚合度寡糖可以被乳酸菌利用,作为益生元很有可能改善肠道菌群的结构。     

Effect of nutrition and Enteromyxum leei infection on gilthead sea bream Sparus aurata intestinal carbohydrate distribution

The effect of a practical plant protein-based diet containing vegetable oils (VO) as the major lipid source on the mucosal carbohydrate pattern of the intestine was studied in gilthead sea bream Sparus aurata challenged with the myxosporean parasite Enteromyxum leei. Fish fed for 9 mo either a fish oil (FO) diet or a blend of VO at 66% of replacement (66VO diet) were exposed to parasite-contaminated water effluent. Samples of the anterior, middle and posterior intestine (AI, MI and PI, respectively) were obtained for parasite diagnosis and histochemistry. Fish were categorised as control (C, not exposed), early (E) or late (L) infected. Mucin and lectin histochemistry was applied to detect the different types of mucins and sialic acid in goblet cells (GC), the brush border and enterocytes. The number of GC stained with periodic acid Schiff (PAS), alcian blue (AB), aldehyde fuchsin-alcian blue (AF-AB), for the detection of neutral, acidic, sulphated and carboxylic mucins, and with the lectin Sambucus nigra agglutinin (SNA), were counted in digital images. The 66VO diet produced a significant decrease of GC with neutral and acidic mucins in the AI and MI, and also of those with carboxylic mucins and sialic acid in the MI. Sulphated mucins and sialic acid were less abundant in the AI than in the MI and PI in the C-66VO treatment. E. leei infection had a strong effect on the number of GC, as E and L infected fish had a significant decrease of GC positive for all the stains versus C fish in PI. Time and diet effects were also observed, since the lowest values were mostly registered in E-66VO fish in PI. In conclusion, though GC depletion was mainly induced by enteromyxosis, an effect of the diet was also observed. Thus, the diet can be a predisposing factor that worsens the disease course.     

Metabolism of 6-nitrochrysene by intestinal microflora.

Since bacterial nitroreduction may play a critical role in the activation of nitropolycyclic aromatic hydrocarbons, we have used batch and semicontinuous culture systems to determine the ability of intestinal microflora to metabolize the carcinogen 6-nitrochrysene (6-NC). 6-NC was metabolized by the intestinal microflora present in the semicontinuous culture system to 6-aminochrysene (6-AC), N-formyl-6-aminochrysene (6-FAC), and 6-nitrosochrysene (6-NOC). These metabolites were isolated and identified by high-performance liquid chromatography, mass spectrometry, and UV-visible spectrophotometry and compared with authentic compounds. Almost all of the 6-NC was metabolized after 10 days. Nitroreduction of 6-NC to 6-AC was rapid; the 6-AC concentration reached a maximum at 48 h. The ratio of the formation of 6-AC to 6-FAC to 6-NOC at 48 h was 93.4:6.3:0.3. Interestingly, compared with results in the semicontinuous culture system, the only metabolite detected in the batch studies was 6-AC. The rate of nitroreduction differed among human, rat, and mouse intestinal microflora, with human intestinal microflora metabolizing 6-NC to the greatest extent. Since 6-AC has been shown to be carcinogenic in mice and since nitroso derivatives of other nitropolycyclic aromatic hydrocarbons are biologically active, our results suggest that the intestinal microflora has the enzymatic capacity to generate genotoxic compounds and may play an important role in the carcinogenicity of 6-NC.     

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.     

Metabolism of anthocyanins and their phenolic degradation products by the intestinal microflora

Anthocyanins are suggested to be responsible for protective effects against cardiovascular diseases and certain forms of cancer. Although previous studies have implicated that intact anthocyanidin glycosides were decreased extensively by interactions in the gastrointestinal tract, only few data are available concerning the metabolism by the intestinal microflora. Using a new in vitro model, we have investigated the microbial deglycosylation and degradation of six anthocyanins exhibiting three different aglycones with mono- or di-beta-D-glycosidic bonds using high-performance liquid chromatography-diode array (HPLC-DAD) and gas chromatography-mass spectrometry (GC-MS) detection. We have found that all anthocyanidin glycosides were hydrolysed by the microflora within 20 min and 2 h of incubation depending on the sugar moiety. Due to the high instability of the liberated aglycones at neutral pH, primary phenolic degradation products were already detected after 20 min of incubation. Further metabolism of the phenolic acids was accompanied by demethylation. Because of their higher chemical and microbial stability, phenolic acids and/or other, not yet identified, anthocyanin metabolites might be mainly responsible for the observed antioxidant activities and other physiological effects in vivo.